dna replication process essay

DNA Replication Steps and Process

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Cell Biology
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DNA replication is the process in which a cell makes an identical copy of its DNA. It is vital for cell growth, repair, and reproduction in organisms as it helps with the transmission of genetic information. The replication process follows several steps involving multiple proteins called replication enzymes and RNA, or ribonucleic acid.

In eukaryotic cells, such as animal cells and plant cells , DNA replication occurs in the S phase of the cell cycle . Before this phase, also known as the synthesis stage, the cell passes through a preparation phase to minimize the chances of errors or mutations being introduced into the new DNA strands.

Key Takeaways

Deoxyribonucleic acid, commonly known as DNA, is a nucleic acid that has three main components: a deoxyribose sugar, a phosphate, and a nitrogenous base.
DNA contains the genetic material for an organism, and it must be copied when a cell divides into daughter cells. The process that copies DNA is called replication.
Replication involves the production of identical helices of DNA from one double-stranded molecule of DNA.
Enzymes are vital to DNA replication since they catalyze very important steps in the process.
The overall DNA replication process is extremely important for both cell growth and reproduction in organisms. It is also vital in the cell repair process.

What Is DNA and Why Does It Replicate?

DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis , biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus , must be replicated to ensure each new cell receives the correct number of chromosomes .

DNA Structure

DNA or deoxyribonucleic acid is a type of molecule known as a nucleic acid . It consists of a five-carbon deoxyribose sugar, a phosphate, and a nitrogenous base. Double-stranded DNA consists of two spiral nucleic acid chains that are twisted into a double helix shape. This twisting allows DNA to be more compact. To fit within the nucleus, DNA is packed into tightly coiled structures called chromatin . Chromatin condenses to form chromosomes during cell division. Before DNA replication, the chromatin loosens, giving cell replication machinery access to the DNA strands.

Replication Preparation and Beginning

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Step 1: Replication Fork Formation

Before DNA can be replicated, the double-stranded molecule must be “unzipped” into two single strands. DNA has four bases called adenine (A), thymine (T), cytosine (C), and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds with guanine. To unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork. This area will be the template for replication to begin.

DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached to the DNA backbone. The 5' end has a phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group attached. This directionality is important for replication as it only progresses in the 5' to 3' direction. However, the replication fork is bi-directional; one strand is oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging strand). The two sides are therefore replicated with two different processes to accommodate the directional difference.

Replication Begins

Step 2: primer binding.

The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase.

DNA Replication: Elongation

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Step 3: Elongation

Enzymes known as DNA polymerases are responsible for creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells . In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV, and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous.

The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.

Step 4: Termination

Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove, and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end, replication produces two DNA molecules, each with one strand from the parent molecule and one new strand.

Replication Enzymes

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DNA replication would not occur without enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include:

DNA helicase: Unwinds and separates double-stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
DNA primase: A type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication.
DNA polymerases: Synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands.
Topoisomerase or DNA Gyrase: Unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.
Exonucleases: Group of enzymes that remove nucleotide bases from the end of a DNA chain.
DNA ligase: Joins DNA fragments together by forming phosphodiester bonds between nucleotides.

DNA Replication Summary

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DNA replication is the production of identical DNA helices from a single double-stranded DNA molecule. Each molecule consists of a strand from the original molecule and a newly formed strand. Prior to replication, the DNA uncoils and strands separate. A replication fork is formed which serves as a template for replication. Primers bind to the DNA and DNA polymerases add new nucleotide sequences in the 5′ to 3′ direction.

This addition is continuous in the leading strand and fragmented in the lagging strand. Once elongation of the DNA strands is complete, the strands are checked for errors, repairs are made, and telomere sequences are added to the ends of the DNA.

An Introduction to DNA Transcription
DNA Definition: Shape, Replication, and Mutation
Understanding the Double-Helix Structure of DNA
How Gene Mutation Works
Genetics Basics
What is Chromatin's Structure and Function?
The Difference Between siRNA and miRNA
Phenotype: How a Gene Is Expressed As a Physical Trait
Genetic Recombination and Crossing Over
What Is Recombinant DNA Technology?
How Chromosome Mutations Occur
Overview of the Stages of Meiosis
A Genetics Definition of Homologous Chromosomes
Types and Examples of DNA Mutations
RFLP and the DNA Analysis Applications
Genes and Genetic Inheritance

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Essay on DNA Replication | Genetics

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In this essay we will discuss about:- 1. Definition of DNA Replication 2. Mechanism of DNA Replication 3. Evidences for Semi-Conservative DNA Replication 4. Models for Replication of Prokaryotic DNA.

Essay # Definition of DNA Replication :

DNA replicates by “unzipping” along the two strands, breaking the hydrogen bonds which link the pairs of nucleotides. Each half then serves as a template for nucleotides available in the cells which are joined together by DNA polymerase. The nucleotides are guanine, cytosine, adenine and thymine. DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule.

This process is important in all known forms of life and the general mechanisms of DNA replication are the same in prokaryotic and eukaryotic organisms. The process by which a DNA molecule makes its identical copies is referred to as DNA replication. In other words, it is the process of duplicating the DNA to make two identical copies. The main points related to DNA replication are briefly presented below.

1. Time of Replication:

The process of DNA replication takes place during cell division. The DNA replication takes place during S sub stage of interphase. In prokaryotes, DNA replication is initiated before the end of the cell cycle. Eukaryotic cells can only initiate DNA replication at the beginning of S phase.

2. Replication Site:

In humans and other eukaryotes, replication occurs in the cell nucleus, whereas in prokaryotes it occurs in the cytoplasm. Prokaryotes have only one active replication site, but eukaryotes have many.

3. Template Used:

The existing DNA is used as a template for the synthesis of new DNA strands. It is possible that during replication on strand of DNA can replicate continuously and the other discontinuously or in piece. The continuously replicating strand is known as leading strand and the discontinuously replicating strand is known as lagging strand.

When one strand of DNA replicates continuously and other discontinuously, it is called semi-discontinuous replication. Earlier it was thought that DNA replicates discontinuously. But now it is believed that DNA replication is semi-discontinuous.

Short segments of nucleotides are synthesized in the lagging strand of DNA as a result of discontinuous replication. These are called Okazaki after the name of discoverer. Okazaki fragments are about 1,500 bases in length in prokaryotes, and 150 bases in eukaryotes

4. Enzymes Involved:

The process of DNA replication takes place under the control of DNA polymerase. In other words, the process is catalized by the polymerase enzyme. In eukaryotes, four types of polymerase enzymes, viz. alpha, delta, gamma and epsilon are used.

DNA Polymerase alpha and delta replicate the DNA. The alpha is associated with initiation, and delta extends the nascent strands. DNA polymerase epsilon and beta are used for repair. DNA polymerase gamma is used for replication of mitochondrial DNA

In prokaryotes [E. coli], there are three major DNA polymerases: DNA polymerase I, II and III. DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication. DNA poly II is exclusively involved in repair. DNA poly III is the major DNA polymerase. All DNA polymerases add to the 3′ OH of the existing polynucleotide.

Currently, six families of polymerases (A, B, C. D, X, Y) have been discovered. At least four different types of DNA polymerases are involved in the replication of DNA in animal cells (POLA, POLG, POLD and POLE).

5. Direction of Replication:

The synthesis of one new strand takes place in 5-3 and that of other in opposite (3-5) direction. The replication may take place either in one direction or in both the directions from the point of origin. When replication proceeds in one direction only, it is called unidirectional replication. When the replication proceeds in both the directions, it is called bidirectional replication.

6. Replication Type:

Based on the direction, the replication may be unidirectional or bidirectional. On the basis of continuity, the replication may be continuous or discontinuous.

7. Origin of Replication:

The point of initiation of DNA replication is known as origin. The progress of replication process is measured from the point of origin.

8. Rate of Replication:

In prokaryotic cells the rate of replication is 500 bases per second. In eukaryotic cells the rate of replication- is 50 bases per second. Eukaryotes have 100 to 3,000 times more DNA than prokaryotes.

9. Replication Models:

There are three models which explain the accurate replication of DNA. These are: (i) dispersive replication, (ii) conservative replication, and (iii) semiconservative replication (Fig. 17.1).

These are explained as follows:

(i) Dispersive Replication:

According to this model of replication the two strands of parental DNA break at several points resulting in several pieces of DNA. Each piece replicates and pieces are reunited randomly, resulting in formation of two copies of DNA from single copy. The new DNA molecules are hybrids which have new and DNA in patches (Fig. 17.2). This method of DNA replication is not accepted as it could not be proved experimentally.

(ii) Conservative Replication:

According to this model of DNA replication two DNA molecules are formed from parental DNA. One copy has both parental strands and the other contains both newly synthesized strands (Fig. 17.2). This method is also not accepted as there is no experimental proof in support of this model.

(iii) Semiconservative Replication:

This model of DNA replication was proposed by Watson and Crick. According to this model of DNA replication, both strands of parental DNA separate from each other. Each old strand synthesizes a new strand. Thus each of the two resulting DNA molecules has one parental and one new strand (Fig. 17.3). This model of DNA replication is universally accepted because there are several evidences in support of this mode.

Essay # Mechanism of DNA Replication :

The semi-conservative model (mechanism) of DNA replication consists of six important steps, viz:

(1) Unwinding,

(2) Binding of RNA primase,

(3) Elongation,

(4) Removal of primers,

(5) Termination, and

(6) DNA repair.

These are briefly discussed as follows:

1. Unwinding:

The first major step in the process of DNA, replication is the breaking of hydrogen bonds between bases of the two anti-parallel strands. The unwinding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T.

That is because there are only two bonds between Adenine and Thymine, whereas there are three hydrogen bonds between Cytosine and Guanine. The Helicase enzyme splits the two strands. The initiation point where the splitting starts is called “origin of replication”. The structure that is created is known as “Replication Fork”.

2. Binding of RNA Primase:

Synthesis of RNA primer is essential for initiation of DNA replication. RNA primer is synthesized by DNA template near the origin with the help of RNA Primase. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3′-5′ strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.

3. Elongation:

The elongation proceeds in both directions, viz. 5′-3′ and 3′-5′ template. The 3′-5′ proceeding daughter strand that uses a 5′-3′ template— is called leading strand because DNA Polymerase ‘a’ can “read” the template and continuously add nucleotides. The 3′-5′ template cannot be “read” by DNA Polymerase a. The replication of this template is complicated and the new strand is called lagging strand.

In the lagging strand the RNA Primase adds more RNA Primers. DNA polymerase a reads the template and lengthens the bubbles. The gap between two RNA primers is called “Okazaki” Fragments. The RNA Primers are necessary for DNA Polymerase a to bind Nucleotides to the 3′ end of them. The daughter strand is elongated with the binding of more DNA nucleotides.

4. Removal of Primers:

The RNA Primers are removed or degraded by DNA polymerase I. This enzyme also catalyzes the synthesis of short DNA segments to replace the primers. The gaps are filled with the action of DNA Polymerase which adds complementary nucleotides to the gaps.

The DNA Ligase enzyme adds phosphate in the remaining gaps of the phosphate-sugar backbone. Each new double helix is consisted of one old and one new chain. This is called semi-conservative replication.

5. Termination:

The termination takes place when the DNA Polymerase reaches to an end of the strands. In other words, it is the separation of replicated linear DNA. After removal of the RNA primer, it is not possible for the DNA Polymerase to seal the gap because there is no primer.

Hence, the end of the parental strand where the last primer binds is not replicated. These ends of linear (chromosomal) DNA consist of noncoding DNA that contains repeat sequences and are called telomeres. A part of the telomere is removed in every cycle of DNA Replication.

6. DNA Repair:

The DNA replication is not completed without DNA repair. The possible errors caused during the DNA replication are repaired by DNA repair mechanism. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps. Similar processes also happen during the steps of DNA Replication of prokaryotes though there are some differences.

Essay # Evidences for Semi-Conservative DNA Replication :

Various experiments have demonstrated the semi-conservative mode of DNA replication. Now it is universally accepted that DNA replicates in a semi-conservative manner. There are three important experiments which support that DNA replication is semi-conservative.

These experiments include:

(1) Meselson and Stahl experiment,

(2) Cairns experiment, and

(3) Taylor’s experiment.

1. Meselson and Stahl Experiment [1958] :

Organism Used:

Meselson and Stahl conducted their experiment with common bacteria of human intestine i.e. Escherichia coli.

They used heavy isotope of nitrogen for labelling DNA. The bacteria were grown on culture medium containing heavy isotope of Nitrogen [N15] for 14 generations (30 minutes per generation) to replace the normal nitrogen [N14] of E. coli with heavy nitrogen.

Then the bacteria were transferred to normal nitrogen medium. The density of DNA was determined after one, two and three generations. Principle Involved. It is possible to detect minute differences in density through density gradient centrifugation. District bands are formed in centrifuge tube for different density DNA.

2. Rolling Circle Model of DNA Replication :

This model of circular DNA replication was proposed in 1968. This model explains mechanism of DNA replication in single stranded circular DNA of viruses, e.g. ɸX174, and the transfer of E. coli sex factor (plasmid). The ϕX174 chromosome consists of a single stranded DNA ring (Positive Strand). This model is most widely accepted.

The mechanism of replication consists of following important steps:

(i) Synthesis of New Strand:

First the chromosome becomes double stranded by synthesis of a negative strand. The original strand is positive. The negative strand is synthesized in side of parental positive strand.

(ii) Cut in Outer Strand:

The negative or inner strand remains a close circle and the positive strand is nicked at a specific site by endonuclease enzyme. This enzyme recognizes a particular sequence at this point. Thus a. linear strand with 3′- and 5′-ends is created.

(iii) Formation of Tail:

The original positive strand comes out in the form of a tail of a single linear strand as a consequence of rolling circle. The 5′-end of the broken strand becomes attached to the plasma membrane of the host bacterium.

Such replicating phage DNA is commonly found associated with bacterial membranes. The unbroken parental strand rolls and unwinds as synthesis proceeds, leaving a ‘tail’ which is attached to the membrane.

(iv) Synthesis of New Strand:

The synthesis of new strand takes place along the parental strand at the tail end in a 3-5 direction. The 3′-end serves as a primer for the synthesis of a new DNA strand under the catalytic action of DNA polymerase. The unbroken strand is used as the template for this purpose, and a complementary strand is synthesized. Thus the parental molecule itself is used as a primer for initiating replication.

New DNA is also synthesized in the tail region in discontinuous segments in the 5-3 direction. This synthesis is presumably preceded by the synthesis of an RNA primer under the catalytic action of RNA polymerase. The tail is cut-off by a specific endonuclease into a unit length progeny rod.

(v) Cutting of Tail:

Now the tail is cut-off into a linear segment by endonuclease. The linear segment becomes circular by joining two ends with the help of ligage enzyme. Thus a new circular molecule is formed which can become new rolling circle and replicate further.

Genetic information is preserved in the single stranded template ring which remains circular and serves as an endless template. There is no swiveling problem or creation of torque in the rolling circle model. As the strands unwind the 3′-end is free to rotate on the unbroken strand. The growing point itself thus serves as a swivel.

Evidence for the rolling circle model has been obtained from the replication of several viruses (M13, P2, T4, λ), replication resulting in transfer of genetic material during mating of bacteria, and the special DNA synthesis during oogenesis in Xenopus.

6 Basic Rules for DNA Replication | Genetics
Replication of DNA: 2 Things to Know About

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Microbe Notes

DNA Replication: Enzymes, Mechanism, Steps, Applications

DNA replication is the process of producing two identical copies of DNA from one original DNA molecule.

DNA is made up of millions of nucleotides, which are composed of deoxyribose sugar, with phosphate and a base.
The complementary pairing of these bases keeps the double strands intact. So, to make two copies of one DNA, these hydrogen bonds in between the bases should be broken to begin replication.
DNA replication is semi-conservative, meaning that each strand in the DNA acts as a template for the synthesis of a new complementary strand. Semi conservative because once DNA molecule is synthesized it has one strand from the parent and the other strand is a newly formed strand.
DNA replication starts by taking one DNA molecule and giving two daughter molecules, with each newly synthesized molecule containing one new and one old strand.
DNA replication simply is the process by which a DNA makes a copy of itself. Though easy as it may sound it’s a complex process happening inside of our cells, and many enzymes, proteins, and metal ions should work coherently to make this process happen.

Table of Contents

Interesting Science Videos

Mechanism of DNA Replication

Summary : DNA replication takes place in three major steps.

Opening of the double-stranded helical structure of DNA and separation of the strands
Priming of the template strands
Assembly of the newly formed DNA segments.
During the separation of DNA, the two strands uncoil at a specific site known as the origin . With the involvement of several enzymes and proteins, they prepare (prime) the strands for duplication.
At the end of the process, DNA polymerase enzyme starts to organize the assembly of the new DNA strands.
These are the general steps of DNA replication for all cells but they may vary specifically, depending on the organism and cell type.
Enzymes play a major role in DNA replication because they catalyze several important stages of the entire process.
DNA replication is one of the most essential mechanisms of a cell’s function and therefore intensive research has been done to understand its processes.
The mechanism of DNA replication is well understood in Escherichia coli, which is also similar to that in eukaryotic cells.
In E.coli, DNA replication is initiated at the oriClocus (oriC), to which DnaA protein binds while hydrolyzing of ATP takes place.

Enzymes and Proteins Used in DNA Replication

A nuclease is an enzyme that can cleave the phosphodiester bonds present in between the nucleotides.
On the basis where they cleave, they are characterized as Exo and endonucleases.
Exonucleases cleave nucleotides from their respective ends. Corresponding to this fact, these exonucleases show activity from both directions 5′ to 3′ and 3′ to 5′.
Endonucleases act on the region in the middle of the targeted nucleotide. They are also endonucleases that are selective to which molecule they cleave and are sub-divided as DNase for DNA for cleaving and RNase for RNA cleaving. Additionally, recently discovered nucleases are also being used for gene editing such as Cas9 in the CRISPR genome editing technique.
Restrictive endonuclease or restriction enzymes are the ones that cleave DNA into fragments at or near the specific recognition sites within the molecule known as restriction sites.
To cleave the DNA, restriction endonuclease makes two incisions, once through each sugar-phosphate backbone of the DNA double helix. These endonucleases recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA.
This specific sequence is usually 4 – 8 bases and is present in the recognition site.
DNA Polymerase

DNA polymerases are the enzyme that is responsible for adding new nucleotides and synthesizing a new strand of DNA by taking the old fragmented strand as a template.
DNA Polymerases also possess exonuclease activity, that cuts incorrectly added nucleotides, and allows the DNA replication to happen without errors.
DNA Polymerase is of many types and functions based on the cell they are found in.
In prokaryotic cells, there are three DNA polymerases: DNA Polymerase Ι, DNA Polymerase ΙΙ and DNA Polymerase ΙΙΙ.
DNA polymerase Ι is a repair polymerase with 5′ to 3′ and 3′ to 5′ exonuclease activity. It is involved in the processing of Okazaki fragments during lagging strand synthesis.
DNA polymerase ΙΙ has 3′ to 5′ exonuclease activity and participated in DNA repair with 5′ to 3′ polymerase activity.
DNA polymerase ΙΙΙ is the primary enzyme involved in the DNA replication of E.coli . It has 3′ to 5′ exonuclease activity and 5′ to 3′ polymerase activity.
In eukaryotic cells, there are five DNA polymerases: DNA Polymerase α, β, γ, δ and ε
DNA polymerase α is a repair polymerase, with 3′ to 5′ exonucleases activities and 5′ to 3′ polymerase activities.
DNA Polymerase β is a repair polymerase.
DNA Polymerase γ shows polymerase activity 5′ to 3′ and exonucleases activity 3′ to 5′, it is involved in Mitochondrial DNA replication
DNA Polymerase δ shows 3′ to 5′ exonuclease activity and 5′ to 3′ polymerase activity. This enzyme is involved in lagging strand synthesis.
DNA Polymerase ε shows 3′ to 5′ and 5′ to 3′ exonucleases activities. This enzyme not only repairs but also synthesizes the leading strand efficiently in a 5′ to 3′ direction. It is the prime enzyme involved in DNA replication.

DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.
This enzyme joins the 3′ hydroxyl group of one nucleotide with the 5′ phosphate end of another nucleotide at an expense of ATP.
DNA helicase
DNA helicase is a motor protein that moves directionally along a nucleic acid phosphodiester backbone, separating two nucleotides of DNA molecule.
They separate double-stranded DNA molecules into single strands allowing each strand to be copied.
During DNA replication, this DNA helicase unwinds DNA at the origin, a site where the replication is to be initiated.
DNA helicase continues to unwind the double helix of DNA and thus forms a structure called replication fork, named after the forked appearance of two strands of DNA when unzipped apart.
It is an energy-driven process as it involves the breaking of Hydrogen bonds between annealed nucleotide bases.
DNA primase
Primase is an enzyme that is capable to synthesize short stretches of RNA sequences known as a primer.
Primers are an integral part of DNA replication. These primers serve as an initiating site for the addition of nucleotides by DNA polymerase.
DNA polymerase can only add nucleotide at pre-existing 3′ Hydroxyl group which is thus provided by the primers.
As we can see that primers are short stretches of RNA, but replication is of DNA, so therefore after elongation of the chains of nucleotides, these primers are replaced by DNA.
DNA topoisomerase
DNA topoisomerase is a class of enzymes that release helical tension during transcription and replication by creating transient nicks within the phosphate backbone on one or both strands of the DNA.
This tension is aroused when the DNA molecule unwinds due to helicase activity and forms a replication fork. The progress of the replication fork generates supercoils, making it hard for other machinery involved to access the DNA molecule.
Class Ι DNA topoisomerase makes a single-stranded break to relax the helix and progress the process.
Class ΙΙ DNA topoisomerase break both the strands of DNA helix, this class of topoisomerases is also very important during the cell cycle for the condensation of chromosomes.
Single strand binding proteins
The single-strand binding (SSB) protein are DNA binding proteins, that binds to single-stranded DNA to facilitate DNA replication.
SSB proteins prevent the hardening of strands during DNA replication. It also protects strands from nuclease degradation and prevents the rewinding of DNA.
These proteins destabilize helical duplexes so that DNA polymerase can hold onto the DNA during DNA replication, recombination, and repair.
It also removes unwanted secondary structures on strands for easy access of the strands to the machinery involved in DNA replication.
Thus, SSB proteins stabilize the single-stranded DNA structure that is important for genomic progression.

So, in summary here are the list of 7 enzymes and proteins used in DNA Replication:

Steps in DNA Replication

Step 1: formation of replication fork.

Before DNA can replicate, this double-stranded molecule must unwind into two single strands to initiate the replication process.
DNA unwinds when the complementary base pairing between the double-stranded is broken, and the site to initiate this unwinding is denoted by specific regions (Adenine and Thymine rich).
These specific coding regions are referred to as Origin of Replication (Ori) and thus the replication process begins.
These origins are targeted by initiator proteins, which go on to recruit more proteins that can help the replication process by forming a replication fork around the Ori.
Within this replication protein complex is an enzyme DNA helicase, which starts to unwind the DNA from its Ori and exposes two strands resembling a Y-like structure referred to as replication fork.
The activity of helicase causes topological stress to the un-winded strand forming supercoiled DNA, this stress is relieved by Topoisomerase by negative supercoiling.
The replication fork is bidirectional; one strand is oriented to 5′ to 3′ direction (leading strand) and the other strand is oriented to 3′ to 5′ direction (lagging strand) but the addition of nucleotide progress only in 5′ to 3′ direction.
The formation of a replication fork exposing two single-stranded strands marks the beginning of Initiation.

Step 2: Initiation

One strand runs from 5′ to 3′ direction towards the replication fork and is referred to as leading strand and the other strand runs from 3′ to 5′ away from the replication fork and is referred to as lagging strands.
To this exposed single-stranded DNA, SSB proteins are adhered to prevent recoiling of DNA and to stabilize it.
After which another enzyme DNA primase comes into action to synthesize a short stretch of RNA primer, which provides a free 3′ hydroxyl group for DNA polymerase can now add nucleotides and extend the new chain of nucleotides.

Step 3: Elongation

Now that primer is added to unzipped two single-stranded DNA, these strands now act as a template for synthesizing new DNAs.
The enzyme DNA polymerase synthesizes new nucleotide to match the template and add on to the free 3′ hydroxyl group provided by the primer in each single-stranded DNA.
The leading strand runs from 5′ to 3′ so the addition of nucleotides by DNA polymerase happens from 5′ to 3′ direction. As the replication fork progresses the addition of nucleotide is continuous thus only requiring the primer once.
However, lagging strands is antiparallel and run from the 5′ to 3′ direction, the continuous addition of nucleotides is not possible as the replication fork progresses, DNA polymerase cannot add complementary nucleotides to the 5′ end. Therefore, multiple primers are required.
Due to this phenomenon, the DNA nucleotides synthesis from lagging strands occurs in fragments. These fragments are termed Okazaki fragments.
Hence, the leading strand using only one primer synthesizes nucleotides continuously, while the lagging strand uses multiple primers and thus synthesizes nucleotides discontinuously.

Step 4: Termination

RNA primers of both leading and lagging strands are cleaved out or degraded by exonucleases activity of DNA polymerase, and the nicks or gaps so formed are filled with DNA and sealed by the enzyme DNA ligase.
DNA polymerase also shows proofreading activity and check, remove and replace any errors.
Interestingly, in eukaryotic organisms having linear DNA, when RNA primer at 5′ end of daughter strand is removed, there is not a preceding 3′ OH such that DNA polymerase can use it to replace with DNA.
So, at the 5′ end of daughter strands, there is a gap (missing DNA). This missing DNA can cause a loss of information contained in that region. This gap must be filled before the next round of replication.
For solving this end replication problem, researchers have found that linear ends of DNA called telomere are used which contain specific G: C rich repeats. These sequences are known as telomere sequences.
These telomere sequences do not code anything but are essential to fill in the gap in the daughter strand and maintain the integrity of DNA.
Eventually, the replication forks terminate at terminating recognizing sequences (ter).
The ter sequences are of around 23 base pairs which facilitate as the binding sites for TUS protein.
This ter- TUS complex arrest replication fork and terminate.

So, in summary, these are the 4 steps of DNA Replication:

Formation of Replication Fork
Termination

Okazaki Fragments

The two DNA strands run in opposite or antiparallel directions, and therefore to continuously synthesize the two new strands at the replication fork requires that one strand is synthesized in the 5’to3′ direction while the other is synthesized in the opposite direction, 3’to 5′.
However, DNA polymerase can only catalyze the polymerization of the dNTPs only in the 5’to 3’direction.
This means that the other opposite new strand is synthesized differently. But how?
By the joining of discontinuous small pieces of DNA that are synthesized backward from the direction of movements of the replication fork. These small pieces or fragments of the new DNA strand are known as the Okasaki Fragments.
The Okasaki fragments are then joined by the action of DNA ligase, which forms an intact new DNA strand known as the lagging strand.
The lagging phase is not synthesized by the primer that initiates the synthesis of the leading strand.
Instead, a short fragment of RNA serves as a primer (RNA primer) for the initiation of replication of the lagging strand.
RNA primers are formed during the synthesis of RNA which is initiated de novo, and an enzyme known as primase synthesizes these short fragments of RNA, which are 3-10 nucleotides long and complementary to the lagging strand template at the replication fork.
The Okazaki fragments are then synthesized by the extension of the RNA primers by DNA polymerase.
However, the newly synthesized lagging strand is that it contains an RNA-DNA joint, defining the critical role of RNA in DNA replication.

Replication Fork Formation and its function

The replication fork is the site of active DNA synthesis, where the DNA helix unwinds and single strands of the DNA replicates.
Several sites of origin represent the replication forks.
The replication fork is formed during DNA strand unwinding by the helicase enzyme which exposes the origin of replication. A short RNA primer is synthesized by primase and elongation done by DNA polymerase.
The replication fork moves in the direction of the new strand synthesis. The new DNA strands are synthesized in two orientations, i.e. 5′ to 3′ direction which is the leading strand, and the 3′ to 5′ orientation which is the lagging strand.
The two sides of the new DNA strand (leading and lagging strand) are replicated in two opposite directions from the replication fork.
Therefore the replication fork is bi-directional.

Leading Strand

The leading strand is the new DNA strand that is continuously synthesized by the DNA polymerase enzyme.
It is the simplest strand that is synthesized during replication.
The synthesis starts after the DNA strand has unzipped and separated. This generates a short piece of RNA known as a primer , by the DNA primase enzyme.
The primer binds to the 5′ end (start) of the strand, thus initiating the synthesize of the new strand (leading strand).
The synthesis of the leading strand is a continuous process.

The Lagging Strand

This is the template strand (3′ to 5′) that is synthesized in a discontinuous manner by RNA primers.
During the synthesis of the leading strand, it exposes small, short strands, or templates that are then used for the synthesis of the Okasaki fragments.
The Okasaki fragments synthesize the lagging strand by the activity of DNA polymerase which adds the pieces of DNA (the Okasaki fragments) to the strand between the primers.
The formation of the lagging strand is a discontinuous process because the newly formed strand (lagging strand) is the fragmentation of short DNA strands.

Applications of DNA Replication

DNA replication makes the transfer of genetic information from one generation to another possible.
It is an important phenomenon happening inside our cells, that allows the body to maintain homeostasis and integrity of the body.
With the available information about DNA replication, scientific communities today have a proper idea of genome sequencing which has now been applied in different expertise ranging from clinical diagnosis to possible treatment of genetic diseases.
DNA replication has made sequencing of whole human genome sequencing possible.
Cloning of genes has also been possible by DNA replication.
Enzymes involved in DNA replication have now been greatly studied due to their wider applications. The recent breakthrough Cas9/ CRISPR technology where nucleases are used to cleave the desired portion of DNA and replace it with required nucleotides is the prime example of how we can use these enzymes and make potential advancements in them thus broadening and exploring their uses.
Polymerase Chain Reaction uses DNA polymerases to repeatedly replicate DNA in-vitro and has numerous applications in diagnosis, sequencing, and recombinant DNA technology.
The formation of complementary DNA (cDNA) can also be considered as an example of a wider application of the enzymes involved in DNA replication.
There are various applications of DNA replication, we can even consider that if there is any technique involving genes, some way or the other DNA replication is applied.

DNA Replication Stress

During DNA replication, the process and the DNA genome undergoes various stress arising from the mechanism. these stresses an result in stalled replication and stalled replication fork formation. Several events contribute to these stresses, including;

Unusual DNA structure
Mismatched ribonucleotides
Tensions arising from concurrent mechanisms of replication and transcription
Inadequate availability of important replication factors
Fragile sites on the replicating DNA strand
Overexpression or constitutive activation of oncogenes
Inaccessible chromatins

Kinase regulatory proteins such as ATM ( ATM serine/threonine kinase ) and ATP are proteins that assist in alleviating replication stress. These proteins get recruited and activated by DNA damages .

Stalled replication forks may collapse if the regulatory proteins do not stabilize, and if and when this happens, initiation of repairing mechanisms to reassembling of the replication fork takes place. this helps to amend damages the damaged ends of DNA.

Similarities between Prokaryotic and Eukaryotic DNA Replication

The unwinding mechanism of DNA before replication is initiated is the same for both Prokaryotes and eukaryotes.
In both organisms, the DNA polymerase enzyme coordinated the synthesis of new DNA strands.
Additionally, both organisms use the semi-conservative replication pattern, making the leading and lagging strands in different directions. Okasaki fragments make the lagging strand.
Lastly, both organisms initiate DNA replication using a short RNA primer.

Eukaryotic vs. Prokaryotic DNA Replication (11 Major Differences)


1.	Occurs in eukaryotic cells.	Occurs in a prokaryotic cell.
2.	This process takes place in the cell’s nucleus.	This process takes place in the cell’s cytoplasm.
3.	There are multiple sites for the origin of replication per DNA molecule.	There is a single site for the origin of replication per DNA molecule.
4.	Initiation of DNA replication is carried out by multi-subunit proteins, origin recognition complex.	Initiation of DNA replication is carried out by protein DnaA and DnaB.
5.	Multiple replication forks are formed in a DNA molecule.	Only two replication forks are formed in a DNA molecule.
6.	Okazaki fragments are short of around 100-200 nucleotides in length	Okazaki fragments are large, around 1000-2000 nucleotides in length.
7.	It is a slow process with around 100 nucleotides added per second.	It is a fast process with around 2000 nucleotides added per second.
8.	DNA is linear and double-stranded.	DNA is circular and double-stranded.
9.	DNA polymerase involved in eukaryotic DNA replication is DNA polymerases ε, α, and δ.	DNA polymerase involved in prokaryotic DNA replication is DNA polymerase Ι, and ΙΙΙ.
10.	Eukaryotic cells have telomeres at the end of DNA thus they are replicated.	Prokaryotic cells have circular DNA thus they are not replicated.
11.	(Telomerase) is needed.	DNA gyrase (Telomerase) is not needed.

Weiguo Cao, “DNA Ligases: Structure, Function, and Mechanism”, Current Organic Chemistry 2002; 6(9). https://doi.org/10.2174/1385272023373950
https://www.nature.com/scitable/definition/helicase-307/
https://proteinswebteam.github.io/interpro-blog/potm/2006_1/Page2.htm
https://www.yourgenome.org/facts/what-is-dna-replication
https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/replication/a/molecular-mechanism-of-dna-replication
https://teachmephysiology.com/biochemistry/cell-growth-death/dna-replication/
https://courses.lumenlearning.com/wm-biology1/chapter/reading-major-enzymes/
https://geneticeducation.co.in/single-stranded-binding-protein-ssb-structure-and-function/
https://www.thoughtco.com/dna-replication-3981005
https://byjus.com/biology/difference-between-prokaryotic-and-eukaryotic-replication/
https://www.vedantu.com/biology/difference-between-prokaryotic-and-eukaryotic-dna-replication
https://www.majordifferences.com/2013/03/difference-between-prokaryotic-and.html

About Author

Rajat Thapa

2 thoughts on “DNA Replication: Enzymes, Mechanism, Steps, Applications”

hello i got a doubt about leading and lagging strand directions. 5` to 3` should be leading strand and 3` to 5` should be a lagging strand. because the polymerase activity goes in 5` to 3` direction so the strand is continuous without any breaks so it is called leading strand and the strand which is in opposite direction to the direction of polymerase is disturbed with breaks hence it is called lagging strand. but you reversed it so please check it and please correct it.

Hello, Yes, you are correct. There has been slight mistakes on the note. We have corrected the note and updated it. Thank you so much for your finding.

9.2 DNA Replication

Learning objectives.

Explain the process of DNA replication
Explain the importance of telomerase to DNA replication
Describe mechanisms of DNA repair

When a cell divides, it is important that each daughter cell receives an identical copy of the DNA. This is accomplished by the process of DNA replication. The replication of DNA occurs during the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis.

The elucidation of the structure of the double helix provided a hint as to how DNA is copied. Recall that adenine nucleotides pair with thymine nucleotides, and cytosine with guanine. This means that the two strands are complementary to each other. For example, a strand of DNA with a nucleotide sequence of AGTCATGA will have a complementary strand with the sequence TCAGTACT ( Figure 9.8 ).

Because of the complementarity of the two strands, having one strand means that it is possible to recreate the other strand. This model for replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied ( Figure 9.9 ).

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. Each new double strand consists of one parental strand and one new daughter strand. This is known as semiconservative replication . When two DNA copies are formed, they have an identical sequence of nucleotide bases and are divided equally into two daughter cells.

DNA Replication in Eukaryotes

Because eukaryotic genomes are very complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination.

Recall that eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. How does the replication machinery know where on the DNA double helix to begin? It turns out that there are specific nucleotide sequences called origins of replication at which replication begins. Certain proteins bind to the origin of replication while an enzyme called helicase unwinds and opens up the DNA helix. As the DNA opens up, Y-shaped structures called replication forks are formed ( Figure 9.10 ). Two replication forks are formed at the origin of replication, and these get extended in both directions as replication proceeds. There are multiple origins of replication on the eukaryotic chromosome, such that replication can occur simultaneously from several places in the genome.

During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3' end of the template. Because DNA polymerase can only add new nucleotides at the end of a backbone, a primer sequence, which provides this starting point, is added with complementary RNA nucleotides. This primer is removed later, and the nucleotides are replaced with DNA nucleotides. One strand, which is complementary to the parental DNA strand, is synthesized continuously toward the replication fork so the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand . Because DNA polymerase can only synthesize DNA in a 5' to 3' direction, the other new strand is put together in short pieces called Okazaki fragments . The Okazaki fragments each require a primer made of RNA to start the synthesis. The strand with the Okazaki fragments is known as the lagging strand . As synthesis proceeds, an enzyme removes the RNA primer, which is then replaced with DNA nucleotides, and the gaps between fragments are sealed by an enzyme called DNA ligase .

The process of DNA replication can be summarized as follows:

DNA unwinds at the origin of replication.
New bases are added to the complementary parental strands. One new strand is made continuously, while the other strand is made in pieces.
Primers are removed, new DNA nucleotides are put in place of the primers and the backbone is sealed by DNA ligase.

Visual Connection

You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

Telomere Replication

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached; however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres , which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase ( Figure 9.11 ) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn ( Figure 9.12 ) received the Nobel Prize for Medicine and Physiology in 2009. Later research using HeLa cells (obtained from Henrietta Lacks) confirmed that telomerase is present in human cells. And in 2001, researchers including Diane L. Wright found that telomerase is necessary for cells in human embryos to rapidly proliferate.

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

DNA Replication in Prokaryotes

Recall that the prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms.

DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes. Table 9.1 summarizes the differences between prokaryotic and eukaryotic replications.

Property	Prokaryotes	Eukaryotes
Origin of replication	Single	Multiple
Rate of replication	1000 nucleotides/s	50 to 100 nucleotides/s
Chromosome structure	circular	linear
Telomerase	Not present	Present

Link to Learning

Click through a tutorial on DNA replication.

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then polymerization continues ( Figure 9.13 a ). Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base ( Figure 9.13 b ). In yet another type of repair, nucleotide excision repair , the DNA double strand is unwound and separated, the incorrect bases are removed along with a few bases on the 5' and 3' end, and these are replaced by copying the template with the help of DNA polymerase ( Figure 9.13 c ). Nucleotide excision repair is particularly important in correcting thymine dimers, which are primarily caused by ultraviolet light. In a thymine dimer, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each other rather than their complementary bases. If the dimer is not removed and repaired it will lead to a mutation. Individuals with flaws in their nucleotide excision repair genes show extreme sensitivity to sunlight and develop skin cancers early in life.

Most mistakes are corrected; if they are not, they may result in a mutation —defined as a permanent change in the DNA sequence. Mutations in repair genes may lead to serious consequences like cancer.

1 Mariella Jaskelioff, et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature , 469 (2011):102–7.

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How Does DNA Replication Occur? What Are The Enzymes Involved?

Structure of dna, directionality, process of replication, termination.

Initiation, elongation and termination are three main steps in DNA replication. Let us now look into more detail of each of them: Step 1: Initiation The point at which the replication begins is known as the Origin of Replication (oriC). Helicase brings about the procedure of strand separation, which leads to the formation of the replication fork. Step 2: Elongation The enzyme DNA Polymerase III makes the new strand by reading the nucleotides on the template strand and specifically adding one nucleotide after the other. If it reads an Adenine (A) on the template, it will only add a Thymine (T). Step 3: Termination When Polymerase III is adding nucleotides to the lagging strand and creating Okazaki fragments, it at times leaves a gap or two between the fragments. These gaps are filled by ligase. It also closes nicks in double-stranded DNA.

We all know that each human being begins their life as a single cell, which divides to form two cells, and these two go on to form four! This process helps us to form our tiny little body, which then grows into an adult! Now while all this is happening, our DNA is also being divided into these cells. But does the cell divide the existing DNA into two parts? Or does it make a second copy? If you think it is the latter, then you are correct! The cell does make a second copy, so when two daughter cells are formed; each one of them gets a complete set of DNA.

Mitosis Vs Meiosis – How Does Cell Division Work?

What Is A Deletion Mutation?

Does Human DNA Change With Time?

What Is Cytokinesis?

Genetic DNA Science Vector Illustration Concept Showing a group of scientist investigating DNA

What Is DNA And How Does It Work?

Chromatin strand and isolated nucleosome( Juan Gaertner)s

Chromatin: Structure And Function Within A Cell

Nuclear Fission v Nuclear Fusion: Differences and Similarities Explained

Germination: How Does A Seed Become A Plant?

Who's J. Robert Oppenheimer And Why Is He A Big Deal?

What are Mutations and what are the different types of Mutations?

Cellular Respiration: How Do Cell Get Energy?

What is Evolution: A REALLY SIMPLE and Brief Explanation

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DNA Replication

Reviewed by: BD Editors

DNA replication is a process that occurs during cellular division where two identical molecules of DNA are created from a single molecule of DNA. As a semiconservative process, a single molecule containing two strands of DNA in double helix formation is separated, where each strand serves as a template for the new DNA molecules. Because the double helix is anti-parallel and DNA polymerase only synthesizes new DNA from 5′-3′, the template strand reading 3′-5′ results in a continuous, leading strand, while the template strand reading 5′-3′ results in a discontinuous, lagging strand. Being a highly regulated process, multiple proteins are required both during and following replication to quickly correct mistakes and damages.

Deoxyribonucleic acid, known simply as DNA, is the blueprint of all living things. DNA contains genes that code for the physical and metabolic information expressed in an individual while having the potential to be passed down to future offspring. Almost all cells have DNA, which is typically stored in the nucleus. Notable cells that lack DNA include anucleate cells (or cells that lack a nucleus, such as red blood cells). Additionally, some cells may still have DNA despite not having a nucleus, such as with bacterial cells.

DNA Nucleotides

DNA is made up of four building block monomers that are known as nucleotides . Nucleotides consists of three groups:

A deoxyribose sugar group
A phosphate group
A nitrogenous base

Adenine nucleotide pairs with thymine and cytosine nucleotide pairing with guanine

All of the nucleotides have the same sugar group and phosphate group, but different nitrogenous bases. The nitrogenous bases in DNA are adenine, guanine, cytosine, and thymine. Adenine and guanine are classified as purines , while cytosine and thymine are classified as pyrimidines . Purines have two rings in their base structures, while pyrimidines have a single ring in their base structures.

(Helpful hint: A simple pneumonic to remember adenine and guanine as purines is “ Pur e A s G old”!)

DNA Structure

Nucleotides are arranged into chains that become individual strands of DNA, which is half of a full DNA molecule. Each strand has a sugar-phosphate backbone that is created when the phosphate of one nucleotide binds to the sugar of the next using a covalent phosphodiester bond . Specifically, the phosphate is found on the 5′ carbon of one nucleotide, while a hydroxyl group (-OH) is found on the 3′ carbon of the next nucleotide’s sugar group. The -OH of the 3′ carbon is removed, where the phosphate group on the 5′ carbon now also bonds to the 3′ carbon.

Three single nucleotides with arrows indicating where the phosphodiester bond will occur

The nitrogenous bases stick out from this backbone. A second DNA strand is matched to this first strand based on complimentary base pairing , where a single purine pairs with a single pyrimidine. Specifically, adenine pairs with thymine and guanine pairs with cytosine. Hydrogen bonds connect the complimentary base pairs, where an adenine- thymine pair has two hydrogen bonds and a guanine-cytosine pair has three hydrogen bonds. A single DNA molecule results in double helix formation when two DNA strands are matched and bonded.

DNA has directionality that can run either 3′-5′ or 5′-3′ based off of the carbons in the sugar group. The two strands of DNA in the double helix must run opposite to each other in an anti-parallel fashion. Therefore, if the first strand starts at the 3′ end and finishes at the 5′ end, then the second strand must run opposite, starting at the 5′ end and finishing at the 3′ end.

3′- AATCGTAA -5′

5′- TTAGCATT -3′

DNA Replication Is Semiconservative

DNA must be fully replicated before cells divide via mitosis to ensure all daughter cells have identical DNA. It was discovered that DNA replication is semiconservative . In semiconservative replication, the double helix splits into two separate strands. During the replication process, an entirely new strand of DNA is created by using the original template strand and matching the complimentary bases.

DNA Replication Process

Proteins in DNA Replication

DNA replication is highly regulated and requires multiple proteins to run efficiently. A majority of these proteins act as stabilizers and enzymes, with enzymes being proteins that behave as catalysts to create and speed up biochemical reactions.

Some of the major proteins in DNA replication include the following:

Helicase : An enzyme that opens the double helix by breaking the hydrogen bonds between complimentary base pairs

Single-strand DNA- binding proteins (SSBPs) : These proteins stabilize the individual strands of DNA to prevent them from reconnecting.

Topoisomerase : Because unwinding of the DNA by helicase creates tension further down the strand, this enzyme relieves tension by making cuts in the DNA and rejoining them before the replication fork arrives.

Primase : An enzyme that adds a primer (which is a short segment of ribonucleic acid, known as RNA) where DNA polymerase III will attach

DNA polymerase III : An enzyme that creates the new DNA strand by adding nucleotides that are complimentary to the template strand

DNA polymerase I : An enzyme that replaces the RNA primer with DNA

DNA ligase : An enzyme that connects the Okazaki fragments on the lagging strand by closing the sugar-phosphate backbone, creating a single DNA strand

Sliding clamp : A protein that holds DNA polymerase III in place

The Replication Bubble

When DNA begins to replicate, a replication bubble is formed that can be detected visually by electron microscopy. A specific sequence of bases- known as the origin of replication – determines where this replication bubble begins. Inside of the bubble, two Y-shaped replication forks result where DNA is actively replicated on either side of the region. The replication forks are formed as the double strands of DNA are separated by helicase in both directions away from the origin of replication. It is at the replication fork that DNA replication proteins attach to fulfill their functions.

Replicating the Leading Strand

As mentioned previously, DNA strands have an anti-parallel nature, where one strand will run 3’-5’ and the other will run opposite from 5’- 3’. DNA polymerase can only synthesize new strands of DNA in the 5’-3’ direction. In order for DNA polymerase to do this, it must read the template strand from 3′-5′. Therefore, replicating the template strand that runs 3’-5’ results in the synthesis of the leading strand . The leading strand is a new strand of DNA that is synthesized in a single, continuous chain that starts at the 5’ end and finishes at the 3’ end.

DNA polymerase continuously on leading strand

DNA replication of the leading strand when the 3’-5’ template strand is used is as follows:

The DNA double helix is opened by helicase into individual strands. Topoisomerase relieves the tension further down the double helix.
SSBPs stabilize the single DNA strands to prevent them from reconnecting.
An RNA primer is added to the leading strand at complimentary bases by primase.
DNA polymerase III attaches to the primer. The sliding clamp stabilizes DNA polymerase III.
DNA polymerase III moves down the leading strand towards the replication fork , adding bases to the new strand from the 5’ end to the 3’ end.

Replicating the Lagging Strand

DNA polymerase can only create new DNA strands from 5’-3’. Therefore, when the 5′-3′ template strand is being replicated- where the new strand must run opposite in the 3′-5′ direction- the new strand cannot be synthesized in a continuous fashion as the leading strand was. To overcome this challenge, additional steps are needed to replicate the 5′-3′ template strand, where this newly synthesized strand is known as the lagging strand .

In order for the lagging strand to be synthesized, DNA needs to be broken down into smaller segments known as Okazaki fragments . Because of these multiple segments, the lagging strand is also known as the discontinuous strand . By creating these multiple segments, DNA polymerase III is able to synthesize a small portion of the new DNA strand away from the replication fork in the correct 5′-3′ direction. As the replication fork continues down the double helix in the 3′ direction of the template strand, another Okazaki fragment can be created closer to the fork. DNA polymerase III binds again to synthesize another portion of the new DNA strand away from the fork until it reaches the previous portion already synthesized. These fragments are then connected, resulting in a single DNA strand. This process continues down the entire length of the DNA.

DNA replication emphasizing the Okazaki fragments needed on the lagging strand

DNA replication of the lagging strand when the 5’-3’ template strand is used is as follows:

SSBPs stabilize the single DNA strands.
Primase adds an RNA primer to the lagging strand.
DNA polymerase III binds to the primer and creates a short segment of newly synthesized DNA from 5′-3′, synthesizing in the opposite direction of the replication fork . This is the first Okazaki fragment.
As helicase further unwinds the double helix and the replication fork moves down the strand, another primer is added closer to the fork. DNA polymerase III attaches to this primer to synthesize a second Okazaki fragment in the 5′-3′ direction away from the replication fork.
Once DNA polymerase III reaches the first Okazaki fragment primer, DNA polymerase I removes the primer and replaces them with the proper complementary bases.
DNA ligase connects the segments of DNA by closing the sugar-phosphate backbone. The two segments are now connected into a single strand.
This process repeats as the replication fork continues down the length of the DNA.

Prokaryotes vs. Eukaryotes

DNA replication overall is fairly conserved across life. However, general differences exist in the enzymes and mechanisms used , as well as time required between species. The largest differences are between the domains of prokaryotes (bacteria and archaea) and eukaryotes (all other plant and animal cells).

Minor differences between these groups include faster replication time in prokaryotes and shorter Okazaki fragments in eukaryotes. Additionally, prokaryotes only have a single origin of replication, while eukaryotes have multiple origins of replication. The most noteworthy difference between these groups however, is that prokaryotes have circular DNA while eukaryotes have linear DNA. Linear eukaryotic DNA creates an additional challenge that must be regulated. This brings us to telomeres.

Chromosome with highlighted red tips to indicate telomeres with closeup of DNA double helix

Because eukaryotic DNA is linear, they have ends that create a challenge. For the leading strand, DNA polymerase III can continue down the entire length of DNA. However, in the lagging strand, a primer must be added in front of the Okazaki fragment being synthesized before DNA polymerase III can attach and synthesize the new DNA strand opposite of the replication fork. Once the last Okazaki fragment is synthesized, a small DNA segment is leftover at the tip of the strand. This segment cannot be left unattended . If this DNA isn’t replicated, then genetic material will be lost each time replication occurs. After several replication cycles, this can result in lost information that could be critical for the individual to survive.

To solve this issue, telomeres are present in eukaryotes. Telomeres are short, repeating segments of DNA that are found at the end of each chromosome and do not contain any coding sequences. These telomeres are synthesized by telomerase , which is an enzyme that contains a short RNA template used to extend the length of the lagging strand. Primers are placed on the telomere where DNA polymerase III can attach to synthesize the final portion of DNA leftover on the lagging strand.

Telomerase adding the RNA template at the end of the DNA

Telomere replication on the lagging strand is as follows:

Telomerase attaches to the very end of the lagging strand, overhanging the unreplicated portion of DNA.
Using its own RNA template, telomerase synthesizes the extending telomere, adding additional bases to the 3’ end of the lagging strand.
Primase adds the primer on the telomere.
DNA polymerase III binds to the primer and moves opposite of telomerase to complete the synthesis of the lagging strand.

Telomeres Affect Cell Age

Telomerase is most commonly active in cell types that divide rapidly, such as with embryonic cells, stem cells, sperm cells, and immune cells. In most other cell types, telomerase activity is turned off, and telomeres become shorter with each DNA replication. This means that cells have a limited number of times that they are able to divide via mitosis before signals are sent to prevent further divisions and DNA damage. As a result, cells have age.

Research has found that increasing telomere length can also increase the lifespan of the cell. While this offers a potential treatment to growth limiting cellular diseases, it also unfortunately assists cancer persistence and survival. Approximately 90% of cancer cells have mutated to turn on telomerase activity in cell types where it should be turned off. This causes another mechanism in which cancer cells can continue to divide without control and become immortalized. Research is ongoing to determine if/how deactivating telomerase activity can either slow or stop cancer progression.

Graphic of chromosomes in normal cells with limited cellular division vs cancer cells and uncontrollable division

DNA Repair and Damage

Incorrect replication.

DNA replication must be fast, but it must also be extremely accurate. DNA replication occurs trillions of times in a single human. Even if there was only a single mistake in each replication, that would add up to trillions of errors that could be detrimental to the individual’s life. So how are mistakes regulated?

The first way this is done is by DNA polymerase proofreading its own work. Each complimentary pair of nucleotides has a distinct shape. Therefore, when the wrong base is placed, the shape is different enough that DNA polymerase can recognize its own mistake. DNA polymerase can then cut out this wrong match and replace it with the correct base.

Cartoon of four repair proteins removing fixing red mistakes on four DNA segments

While DNA polymerase is able to proofread its own work, sometimes mistakes still goes amiss. Once DNA polymerase continues down the length of the strand, mismatch repair proteins are able to edit any additional mistakes. By using markers on the old strand of DNA, the mismatch repair proteins can distinguish sequence errors on the new strand. They then remove the mismatched nucleotide and replace it accordingly. With both DNA polymerase proofreading and the mismatch repair proteins correcting additional mistakes, there is roughly only one mistake for every 1 billion nucleotides synthesized.

Environmental Damage

Environmental factors- such a UV radiation, X-rays, and chemical exposure- can damage DNA. For example, UV radiation found in sunlight and tanning booths can create a thymine dimer where two thymine bases next to each other form a covalent bond. This thus creates a bump in the DNA strand that prevents DNA polymerase from synthesizing past this point. These circumstances can become detrimental, and systems must be put into place to repair damages such as this.

In the case of the UV radiation, eukaryotic cells have adapted a nucleotide excision repair system that is able to detect deformities in the shape of the DNA helix. At least 18 different proteins work together to remove this deformity, using the non-damaged strand as a template to repair the damaged strand. Prokaryotic cells have a simpler but similar nucleotide excision repair system that only requires three proteins. However, for UV radiation specifically, prokaryotes use an enzyme known as photolyase to detect this damage and make repairs.

Normal DNA pointing to damaged DNA with thymine dimer after UV radiation

DNA replication is a highly regulated molecular process where a single molecule of DNA is duplicated to result in two identical DNA molecules. As a semiconservative process, the double helix is broken down into two strands, where each strand serves as the template for the newly synthesized strand by matching complementary bases. Because DNA polymerase III can only synthesize the new strands from 5′-3′, this results in a leading strand that is continuously synthesized and a lagging strand that requires the use of Okazaki fragments. Meanwhile, because eukaryotes have linear DNA, telomeres are needed to ensure genetic information is not lost during replication. Because DNA is critical to life, research continues to better understand and treat diseases caused by mutations and damages in an individual’s DNA.

1. In double-stranded DNA, which nucleotide does adenine pair with?

2. What direction does DNA polymerase synthesize new DNA strands?

3. When the leading strand is being synthesized, what direction is the template strand?

4. When the lagging strand is being synthesized, what direction is the template strand?

5. DNA polymerase synthesizes new strands by matching complimentary base pairs from an external DNA template strand. There is not an external template for telomerase to use when synthesizing telomeres however. How does telomerase recognize what bases to add to the lagging strand and where to start?

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Bibliography

Freeman, S., Quillin, K., Allison, L. A., Black, M., Podgorski, G., Taylor, E., & Carmichael, J. (2017). “Biological science (Sixth edition.).” Boston: Pearson Learning.
Miesfeld, R. & McEvoy, M. (2017). “Biochemistry (Preliminary Edition).” New York: W.W. Norton & Company.
Srinivas, N., Rachakonda, S., & Kumar, R. (2020). “Telomeres and Telomere Length: A General Overview.” Cancers , 12 (3), 558.

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Chapter 16 | DNA, RNA and Proteins

By the end of this section, you will be able to:

Explain the process of DNA replication in prokaryotes
Discuss the role of different enzymes and proteins in supporting this process

DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.

How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be only extended in this direction). It also requires a free 3′-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3′-OH end. Another enzyme, RNA primase , synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer . DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand ( Figure 16.15 ).

Illustration shows the replication fork. Helicase unwinds the helix, and single-strand binding proteins prevent the helix from re-forming. Topoisomerase prevents the DNA from getting too tightly coiled ahead of the replication fork. DNA primase forms an RNA primer, and DNA polymerase extends the DNA strand from the RNA primer. DNA synthesis occurs only in the 5' to 3' direction. On the leading strand, DNA synthesis occurs continuously. On the lagging strand, DNA synthesis restarts many times as the helix unwinds, resulting in many short fragments called “Okazaki fragments.” DNA ligase joins the Okazaki fragments together into a single DNA molecule.

The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. One strand, which is complementary to the 3′ to 5′ parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand . The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments , each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand .

The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to 3′. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in by deoxyribonucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.

Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows:

DNA unwinds at the origin of replication.
Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
Primase synthesizes RNA primers complementary to the DNA strand.
DNA polymerase starts adding nucleotides to the 3′-OH end of the primer.
Elongation of both the lagging and the leading strand continues.
RNA primers are removed by exonuclease activity.
Gaps are filled by DNA pol by adding dNTPs.
The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.

Table 16.1 summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.

Prokaryotic DNA Replication: Enzymes and Their Function



DNA pol I	Exonuclease activity removes RNA primer and replaces with newly synthesized DNA
DNA pol II	Repair function
DNA pol III	Main enzyme that adds nucleotides in the 5′-3′ direction
Helicase	Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase	Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase	Synthesizes RNA primers needed to start replication
Sliding Clamp	Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase	Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA
Single-strand binding proteins (SSB)	Binds to single-stranded DNA to avoid DNA rewinding back.

Review the full process of DNA replication here.

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CIE A-level Biology Revision Notes

DNA Replication (A-level Biology)

Why does dna replicate.

Most organisms produce new cells every day through a process called cell division which occurs continuously.

Table of Contents

DNA replication occurs before the cell divides. DNA replicates itself during the S phase of the cell cycle so that each daughter cells has a copy of the DNA after cell division.

DNA replication mean that parents can pass their DNA to their offspring. This passing of DNA and the genetic information stored in DNA is known as “ Genetic Continuity ”. The replication of DNA is crucial to ensuring genetic continuity both during cell division and between parents and offspring during reproduction.

The Process of DNA Replication

1) double helix unwinding.

The first step of DNA replication is unwinding of the DNA double helix . Because DNA is a base-paired double helix, it replicates itself by unwinding and using each of its strands as a template to form a new strand.
Hydrogen bonds are broken during unwinding . There is breakage of hydrogen bonds between complementary base pairs on the two polynucleotide chains.
An enzyme called DNA helicase is involved . DNA helicase unwinds the DNA by breaking the hydrogen bonds between complementary base pairs on the two strands of DNA.
It is important to understand that the entire DNA does not unwind simultaneously . DNA replication occurs along an entire molecule of DNA and the unwinding happens in one region of the molecule at a time. This is done to ensure stability of the molecule.
The unwound region of the DNA is called a “ replication fork ” . DNA gets unwound in one direction only, meaning the replication fork moves continuously in a unilateral direction.

2) Semi-Conservative Replication

DNA replication is semiconservative . The original strands of DNA act as a template for the synthesis of new strands of DNA. So each new DNA molecule is made up of one parent strand (see next point) from the original DNA molecule, and one new, daughter strand.

The unwound strands of DNA are referred to as the parental strands . Free floating nucleotides in the nucleus are attracted to these parental strands of DNA.

3) DNA Polymerase (Condensation Reactions)

Condensation reactions occur to complete DNA replication . The newly attracted nucleotides are only hydrogen bonded with the parental strand. To create a new strand of DNA, condensation reactions between these nucleotides need to occur in order to synthesise the daughter polynucleotide chain in order to complete DNA replication.
DNA polymerase is the key enzyme. These condensation reactions are catalysed by the enzyme DNA polymerase , which reads the nucleotides and enables them to join. DNA ligase is responsible for the actual condensation reaction.

Mechanism of DNA Polymerase

In a DNA double helix, the two strands are antiparallel. We previously established how in DNA, one strands goes from 3’ to 5’, and the opposite strand goes from 5’ to 3’.

DNA polymerase Works in the 5′ to 3′ direction

DNA polymerase catalyses addition of free nucleotides . DNA polymerase “reads” the parental strand, and catalyses the addition of the free-floating nucleotides.
DNA polymerase starts building at the 5’ end of the daughter strand . DNA polymerase can only bind to the 3′ end of a parental strand and work in one direction. This means they build the new strand in the 5′ to 3′ direction only.
One of the daughter strands will be the leading strand. Since DNA strands are antiparallel but DNA polymerase can only work in one direction, replication has to occur in opposite directions on the two strands. Remember that DNA is also being unwound in one direction only too. The daughter strand which will go in the 5′ to 3′ direction towards the replication fork can be made continuously because the DNA polymerase can move continuously in this direction and follow the replication fork. This strand is called the leading strand .
The other daughter strand will be the lagging strand. The other daughter strand will run 5′ to 3′ away from the replication fork. This strand cannot be made continuously as DNA polymerase can only build the new strand in opposite direction. Thus, DNA polymerase will need to keep detaching and reattaching to this strand, and this results in the new strand being built in short segments. This strand is called the lagging strand .

DNA polymerase reads and DNA ligase catalyses

DNA polymerase reads the nucleotide sequence . When DNA polymerase binds to the parental DNA it reads the nucleotide sequence and recruits complementary nucleotides to form a hydrogen bond with the parental nucleotide. In doing so, DNA polymerase carries out a “proofreading” activity. It makes sure that only complementary nucleotides are pairing in order to prevent mutations from happening.
DNA ligase catalyses condensation reactions . As the DNA polymerase recruits new nucleotides, DNA ligase catalyses condensation reactions between the new nucleotides to create a polynucleotide chain.

DNA Replication is the process of making a copy of the genetic information contained in DNA. This process is necessary for cell division and the transfer of genetic information from one generation to the next.

DNA Replication occurs through the semi-conservative mechanism, where each strand of the DNA double helix acts as a template for the synthesis of a new complementary strand. The DNA strands separate, and each strand is used as a template to build a new complementary strand by the addition of nucleotides.

The main enzymes involved in DNA Replication are helicase, primase, DNA polymerase, and ligase. helicase unwinds the double helix, primase synthesizes RNA primers, DNA polymerase adds nucleotides to the template strand, and ligase seals the gaps between the nucleotides.

RNA primers are short stretches of RNA that are synthesized by primase and are used to initiate DNA Replication. The primers provide a starting point for the addition of nucleotides by DNA polymerase. Once the primer is in place, the DNA polymerase can start adding nucleotides to the template strand, building the new complementary strand.

DNA Replication ensures the accuracy of the copied genetic information through the proofreading function of DNA polymerase. DNA polymerase checks each nucleotide before adding it to the new strand and corrects any mistakes. Additionally, there are enzymes, such as exonucleases, that can remove incorrect nucleotides from the new strand before the replication process is complete.

If DNA Replication goes wrong, it can result in mutations in the genetic information. Mutations can have a variety of effects on an organism, ranging from no effect at all to serious health problems. Some mutations can lead to the development of diseases, such as cancer, while others can result in changes in physical characteristics or behavior.

DNA Replication is important because it allows for the transfer of genetic information from one generation to the next. It is also necessary for cell division, allowing cells to divide and form new cells. Additionally, DNA Replication is essential for the repair of damaged DNA, helping to maintain the stability and integrity of the genetic information.

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CIE 1 Cell structure

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biology), sources of atp during contraction (a-level biology), the ultrastructure of the sarcomere during contraction (a-level biology), the role of troponin and tropomyosin (a-level biology), the structure of myofibrils (a-level biology), slow and fast twitch muscles (a-level biology), the structure of mammalian muscles (a-level biology), how muscles allow movement (a-level biology), the neuromuscular junction (a-level biology), features of synapses (a-level biology), cie 16 inherited change, calculating genetic diversity (a-level biology), how meiosis produces variation (a-level biology), cell division by meiosis (a-level biology), importance of meiosis (a-level biology), cie 17 selection and evolution, types of selection (a-level biology), mechanism of natural selection (a-level biology), types of variation (a-level biology), cie 18 biodiversity, classification and conservation, biodiversity and gene technology (a-level biology), factors affecting biodiversity (a-level biology), 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biology), enzymes: key concepts (a-level biology), enzymes: introduction (a-level biology), cie 4 cell membranes and transport, transport across membranes: active transport (a-level biology), investigating transport across membranes (a-level biology), transport across membranes: osmosis (a-level biology), transport across membranes: diffusion (a-level biology), signalling across cell membranes (a-level biology), function of cell membrane (a-level biology), factors affecting cell membrane structure (a-level biology), structure of cell membranes (a-level biology), cie 5 the mitotic cell cycle, chromosome mutations (a-level biology), cell division: checkpoints and mutations (a-level biology), cell division: phases of mitosis (a-level biology), cell division: the cell cycle (a-level biology), cell division: chromosomes (a-level biology), cie 6 nucleic acids and protein synthesis, transfer rna (a-level biology), transcription (a-level biology), messenger rna (a-level biology), introducing the genetic code (a-level biology), genes and protein synthesis (a-level biology), synthesising proteins from dna (a-level biology), structure of rna (a-level biology), dna structure and the double helix (a-level biology), polynucleotides (a-level biology), cie 7 transport in plants, translocation and evidence of the mass flow hypothesis (a-level biology), the phloem (a-level biology), importance of and evidence for transpiration (a-level biology), introduction to transpiration (a-level biology), the pathway and movement of water into the roots and xylem (a-level biology), the xylem (a-level biology), cie 8 transport in mammals, controlling heart rate (a-level biology), structure of the heart (a-level biology), transport of carbon dioxide (a-level biology), transport of oxygen (a-level biology), exchange in capillaries (a-level biology), structure and function of blood vessels (a-level biology), cie 9 gas exchange and smoking, lung disease (a-level biology), pulmonary ventilation rate (a-level biology), ventilation (a-level biology), structure of the lungs (a-level biology), general features of exchange surfaces (a-level biology), understanding surface area to volume ratio (a-level biology), the need for exchange surfaces (a-level biology), edexcel a 1: lifestyle, health and risk, phospholipids – introduction (a-level biology), edexcel a 2: genes and health, features of the genetic code (a-level biology), gas exchange in plants (a-level biology), gas exchange in insects (a-level biology), edexcel a 3: voice of the genome, edexcel a 4: biodiversity and natural resources, edexcel a 5: on the wild side, reducing biomass loss (a-level biology), sources of biomass loss (a-level biology), transfer of biomass (a-level biology), measuring biomass (a-level biology), net primary production (a-level biology), gross primary production (a-level biology), trophic levels (a-level biology), edexcel a 6: immunity, infection & forensics, microbial techniques (a-level biology), the innate immune response (a-level biology), edexcel a 7: run for your life, edexcel a 8: grey matter, inhibitory synapses (a-level biology), synaptic transmission (a-level biology), the structure of the synapse (a-level biology), factors affecting the speed of transmission (a-level biology), myelination (a-level biology), the refractory period (a-level biology), all or nothing principle (a-level biology), edexcel b 1: biological molecules, inorganic ions (a-level biology), edexcel b 10: ecosystems, nitrogen cycle: nitrification and denitrification (a-level biology), the phosphorus cycle (a-level biology), nitrogen cycle: fixation and ammonification (a-level biology), introduction to nutrient cycles (a-level biology), edexcel b 2: cells, viruses, reproduction, edexcel b 3: classification & biodiversity, edexcel b 4: exchange and transport, edexcel b 5: energy for biological processes, edexcel b 6: microbiology and pathogens, edexcel b 7: modern genetics, edexcel b 8: origins of genetic variation, edexcel b 9: control systems, ocr 2.1.1 cell structure, structure of prokaryotic cells (a-level biology), eukaryotic cells: comparing plant and animal cells (a-level biology), eukaryotic cells: plant cell organelles (a-level biology), eukaryotic cells: the endoplasmic reticulum (a-level biology), eukaryotic cells: the golgi apparatus and lysosomes (a-level biology), ocr 2.1.2 biological molecules, introduction to eukaryotic cells and organelles (a-level biology), ocr 2.1.3 nucleotides and nucleic acids, ocr 2.1.4 enzymes, ocr 2.1.5 biological membranes, ocr 2.1.6 cell division, diversity & organisation, ocr 3.1.1 exchange surfaces, ocr 3.1.2 transport in animals, ocr 3.1.3 transport in plants, examples of xerophytes (a-level biology), introduction to xerophytes (a-level biology), ocr 4.1.1 communicable diseases, structure of viruses (a-level biology), ocr 4.2.1 biodiversity, ocr 4.2.2 classification and evolution, ocr 5.1.1 communication and homeostasis, the resting 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Semi-Conservative DNA Replication: Meselson and Stahl

This structure has novel features which are of considerable biological interest . . . It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material . —Watson & Crick (1953)

Perhaps the most significant aspect of Watson and Crick's discovery of DNA structure was not that it provided scientists with a three-dimensional model of this molecule , but rather that this structure seemed to reveal the way in which DNA was replicated. As noted in their 1953 paper, Watson and Crick strongly suspected that the specific base pairings within the DNA double helix existed in order to ensure a controlled system of DNA replication . However, it took several years of subsequent study, including a classic 1958 experiment by American geneticists Matthew Meselson and Franklin Stahl, before the exact relationship between DNA structure and replication was understood.

Three Proposed Models for DNA Replication

Replication is the process by which a cell copies its DNA prior to division. In humans, for example, each parent cell must copy its entire six billion base pairs of DNA before undergoing mitosis . The molecular details of DNA replication are described elsewhere, and they were not known until some time after Watson and Crick's discovery. In fact, before such details could be determined, scientists were faced with a more fundamental research concern. Specifically, they wanted to know the overall nature of the process by which DNA replication occurs.

Defining the Models

Semiconservative replication was not the only model of DNA replication proposed during the mid-1950s, however. In fact, two other prominent hypotheses were put also forth: conservative replication and dispersive replication. According to the conservative replication model, the entire original DNA double helix serves as a template for a new double helix, such that each round of cell division produces one daughter cell with a completely new DNA double helix and another daughter cell with a completely intact old (or original) DNA double helix. On the other hand, in the dispersive replication model, the original DNA double helix breaks apart into fragments, and each fragment then serves as a template for a new DNA fragment. As a result, every cell division produces two cells with varying amounts of old and new DNA (Figure 1).

Making Predictions Based on the Models

Meselson and stahl’s elegant experiment.

The duo thus began their experiment by choosing two isotopes of nitrogen—the common and lighter 14 N, and the rare and heavier 15 N (so-called "heavy" nitrogen)—as their labels and a technique known as cesium chloride (CsCl) equilibrium density gradient centrifugation as their sedimentation method. Meselson and Stahl opted for nitrogen because it is an essential chemical component of DNA; therefore, every time a cell divides and its DNA replicates, it incorporates new N atoms into the DNA of either one or both of its two daughter cells, depending on which model was correct. "If several different density species of DNA are present," they predicted, "each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way, DNA labeled with heavy nitrogen ( 15 N) may be resolved from unlabeled DNA" (Meselson & Stahl, 1958).

The scientists then continued their experiment by growing a culture of E. coli bacteria in a medium that had the heavier 15 N (in the form of 15 N-labeled ammonium chloride) as its only source of nitrogen. In fact, they did this for 14 bacterial generations, which was long enough to create a population of bacterial cells that contained only the heavier isotope (all the original 14 N-containing cells had died by then). Next, they changed the medium to one containing only 14 N-labeled ammonium salts as the sole nitrogen source. So, from that point onward, every new strand of DNA would be built with 14 N rather than 15 N.

Just prior to the addition of 14 N and periodically thereafter, as the bacterial cells grew and replicated, Meselson and Stahl sampled DNA for use in equilibrium density gradient centrifugation to determine how much 15 N (from the original or old DNA) versus 14 N (from the new DNA) was present. For the centrifugation procedure, they mixed the DNA samples with a solution of cesium chloride and then centrifuged the samples for enough time to allow the heavier 15 N and lighter 14 N DNA to migrate to different positions in the centrifuge tube.

By way of centrifugation, the scientists found that DNA composed entirely of 15 N-labeled DNA (i.e., DNA collected just prior to changing the culture from one containing only 15 N to one containing only 14 N) formed a single distinct band, because both of its strands were made entirely in the "heavy" nitrogen medium. Following a single round of replication, the DNA again formed a single distinct band, but the band was located in a different position along the centrifugation gradient. Specifically, it was found midway between where all the 15 N and all the 14 N DNA would have migrated—in other words, halfway between "heavy" and "light" (Figure 2). Based on these findings, the scientists were immediately able to exclude the conservative model of replication as a possibility. After all, if DNA replicated conservatively, there should have been two distinct bands after a single round of replication; half of the new DNA would have migrated to the same position as it did before the culture was transferred to the 14 N-containing medium (i.e., to the "heavy" position), and only the other half would have migrated to the new position (i.e., to the "light" position). That left the scientists with only two options: either DNA replicated semiconservatively, as Watson and Crick had predicted, or it replicated dispersively.

Straight or Circular?

Following publication of Meselson and Stahl's results, many scientists confirmed that semiconservative replication was the rule, not just in E. coli , but in every other species studied as well. To date, no one has found any evidence for either conservative or dispersive DNA replication. Scientists have found, however, that semiconservative replication can occur in different ways—for example, it may proceed in either a circular or a linear fashion, depending on chromosome shape.

In fact, in the early 1960s, English molecular biologist John Cairns performed another remarkably elegant experiment to demonstrate that E. coli and other bacteria with circular chromosomes undergo what he termed " theta replication ," because the structure generated resembles the Greek letter theta (Θ). Specifically, Cairns grew E. coli bacteria in the presence of radioactive nucleotides such that, after replication, each new DNA molecule had one radioactive (hot) strand and one nonradioactive strand. He then isolated the newly replicated DNA and used it to produce an electron micrograph image of the Θ-shaped replication process (Figure 3; Cairns, 1961).

References and Recommended Reading

Cairns, J. The bacterial chromosome and its manner of replication as seen by autoradiography. Journal of Molecular Biology 6 , 208–213 (1961)

Meselson, M., & Stahl, F. The replication of DNA in Escherichia coli . Proceedings of the National Academy of Sciences 44 , 671–682 (1958)

Watson, J. D., & Crick, F. H. C. A structure for deoxyribose nucleic acid. Nature 171 , 737–738 (1953) ( link to article ).

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Dna Replication Machinery Enzymes

DNA Replication Process - Machinery And Enzymes

We know that DNA is a self-replicating structure and DNA replicates semi-conservatively. However, DNA replication is catalyzed by a set of enzymes. Let’s learn about the DNA replication process and the role of enzymes involved in DNA replication.

DNA Replication

In the process of DNA replication, the DNA makes multiple copies of itself. It is a biological polymerisation, which proceeds in the sequence of initiation, elongation, and termination. It is an enzyme-catalysed reaction. DNA Polymerase is the main enzyme in the replication process.

DNA Replication Process

DNA Replication Steps

Following are the important steps involved in DNA replication:

DNA replication demands a high degree of accuracy because even a minute mistake would result in mutations. Thus, replication cannot initiate randomly at any point in DNA.

For the replication to begin there is a particular region called the origin of replication. This is the point where the replication originates. Replication begins with the spotting of this origin followed by the unwinding of the two DNA strands.

Unzipping of DNA strands in their entire length is not feasible due to high energy input. Hence, first, a replication fork is created catalysed by the helicase enzyme, which unzips the DNA strand.

As the strands are separated, the polymerase enzymes start synthesising the complementary sequence in each of the strands. The parental strands will act as a template for newly synthesising daughter strands.

It is to be noted that elongation is unidirectional i.e. DNA is always polymerised only in the 5′ to 3′ direction. Therefore, in one strand (the template 3 ‘ →5 ‘ ) it is continuous, hence called continuous replication while on the other strand (the template 5 ‘ →3 ‘ ) it is discontinuous replication. They occur as fragments called Okazaki fragments. The enzyme called DNA ligase joins them later.

DNA Replication Fork

Termination

Termination of replication occurs in different ways in different organisms. In E.coli like organisms, chromosomes are circular. And this happens when the two replication forks between the two terminals meet each other.

Role of Enzymes in DNA Replication

DNA replication is a highly enzyme-dependent process. There are many enzymes involved in DNA replication, which includes the enzymes, DNA-dependent DNA polymerase, helicase, ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.

DNA-dependent DNA polymerase

It helps in the polymerisation, catalyses and regularises the whole process of DNA replication with the support of other enzymes. Deoxyribonucleoside triphosphates are the substrate as well as the energy provider for the replication process. DNA polymerase is of three types:

DNA Polymerase I

It is a DNA repair enzyme. It is involved in three activities:

5′-3′ polymerase activity
5′-3′ exonuclease activity
3′-5′ exonuclease activity

DNA Polymerase II

It is responsible for primer extension and proofreading.

DNA Polymerase III

It is responsible for in vivo DNA replication.

Helicase is the enzyme, which unzips the DNA strands by breaking the hydrogen bonds between them. Thus, it helps in the formation of the replication fork.

Ligase is the enzyme which joins together the Okazaki fragments of the discontinuous DNA strands.

This enzyme helps in the synthesis of RNA primer complementary to the DNA template strand.

Endonucleases

These produce a single-stranded or a double-stranded cut in a DNA molecule.

Single-stranded Binding Proteins

It binds to single-stranded DNA and protects it from forming secondary structures.

Also Read: Difference between Replication and Transcription

DNA Replication Process in Prokaryotes

The DNA replication in prokaryotes takes place in the following place:

The two strands of DNA unwind at the origin of replication.
Helicase opens the DNA and replication forks are formed.
The DNA is coated by the single-strand binding proteins around the replication fork to prevent rewinding of DNA.
Topoisomerase prevents the supercoiling of DNA.
RNA primers are synthesised by primase. These primers are complementary to the DNA strand.
DNA polymerase III starts adding nucleotides at the end of the primers.
The leading and lagging strands continue to elongate.
The primers are removed and the gaps are filled with DNA Polymerase I and sealed by ligase.

DNA Replication in Eukaryotes

The DNA replication in eukaryotes is similar to the DNA replication in prokaryotes. However, the initiation process is more complex in eukaryotes than prokaryotes. In eukaryotes, there are multiple origins of replication present. A pre-replication complex is made with other initiator proteins. The process is entirely the same but the enzymes used are different. E.g. in eukaryotes, the polymerisation process is carried out by the enzyme Pol δ, whereas in prokaryotes it is done by DNA Pol III.

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DNA Replication as a Semiconservative Process Essay

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The process of DNA replication has been studied extensively as the pathway to understanding the processes of inheritance and the possible platform for addressing a range of health issues occurring as a result of DNA mutations.

However, the subject matter is still plagued by grey areas that require further analysis, the very properties of the process being one of the core issues of debating. Specifically, whether DNA replication can be deemed as semiconservative remains largely an unanswered question (Georgia Highlands College, n.d.). I believe that, despite the lack of certainty regarding the problem under analysis, it would be reasonable to believe that DNA replication is semiconservative since it is consistent with the fact that, during the reproduction process, DNS is separated into two bands.

The statement concerning DNA replication being a semiconservative process that leads to the development of two separate strands of DNA material has been supported by a vast range of evidence. Recent experiments point to the correctness of the semiconservative framework as the most legitimate theory that allows describing the process of DNA replication in the greatest detail possible (Georgia Highlands College, n.d.). In order to concede that the process of DNA replication is semiconservative, one should take a closer look at the outcomes of the experiment performed.

Since the test performed by Meselson and Stahl showed that the amount of the DNA material was equal in two daughter cells, yet the density thereof was different, the presence of semiconservative properties in DNA replication can be regarded as proven. By asserting that the observed tendency could be found in not only the strands of E.coli but also in other species, Meselson and Stahl made it evident that the DNA replication did, in fact, show semiconservative properties (Georgia Highlands College, n.d.). Thus, I insist that the existing evidence points to the DNA replication process being semiconservative.

The described outcomes of the experiment also lead to a vast range of conclusions concerning the nature and outcomes of DNA replications in different species. By defining DNA replication as semiconservative, the researchers made it evident that every double helix axis in the DNA structure built with the help of DNA polymerases leads to the creation of an entirely new strand that acts as complementary (Georgia Highlands College, n.d.).

It should also be borne in mind that the specified characteristic of the DNA structure makes it possible for the new strand, which is also known as the leading one, to emerge as a continuous piece, whereas the complementary one, or the lagging strand, occurs as a combination of smaller pieces (Georgia Highlands College, n.d.). By applying the notion of the DNA replication process being semiconservative, one can explain the observed changes within the DNA framework and provide the foundation for the further analysis of the subject matter.

Indeed, the results of the experiment described above cannot be deemed as consistent with the theory of dispersive replication, which has been offered as the alternative to the semiconservative framework. Using isotopes of nitrogen as the tools for labeling the DNA of the studied bacteria, Meselson and Stahl staged an experiment in the course of which the nature of the DNA replication process and the basis for its implementation were studied (Georgia Highlands College, n.d.). The semiconservative assumption made by the scientists implied that, in the process of replication, entirely new strands of DNA were produced with the help of the ones that were already present in the cells of the bacteria under analysis.

During the experiment, it was discovered that each of the nitrogenous bases presented in the DNA structure is only capable of connecting to its corresponding complementary partner. For adenine, the process of pairing occurs with thymine, whereas cytosine is connected to guanine in the process (Georgia Highlands College, n.d.). The resulting replication process, thus, takes place due to the combination of the helicase and DNA polymerase procedures (Georgia Highlands College, n.d.). Therefore, I strongly believe that the principle of DNA replication as the notion based on the semiconservative framework seems to be quite valid, given the vast amount of supportive evidence that has been collected.

Based on the outcomes of the Meselson–Stahl experiment, during which the DNA showed a strong propensity toward splitting into two distinct brands, the assumption that the DNA process is semiconservative can be regarded as confirmed. Even though it could be alleged that the current research has been erroneous and that the process of DNA replication may involve different processes and be based on an entirely different principle, the veracity of the identified statement is quite feeble. Thus, the stages of DNA replication can be seen as the semiconservative process. The presence of a synthesized strand along with the preexisting template one has proven to be the most sensible way of looking at the DNA replication stage.

Georgia Highlands College. (n.d.). Chapter 14 – DNA structure and function . Web.

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IvyPanda. (2021, June 2). DNA Replication as a Semiconservative Process. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/

"DNA Replication as a Semiconservative Process." IvyPanda , 2 June 2021, ivypanda.com/essays/dna-replication-as-a-semiconservative-process/.

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IvyPanda . 2021. "DNA Replication as a Semiconservative Process." June 2, 2021. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/.

1. IvyPanda . "DNA Replication as a Semiconservative Process." June 2, 2021. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/.

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v.4(1); 2023 Feb

DNA replication: Mechanisms and therapeutic interventions for diseases

Hao‐yun song.

1 School of Basic Medical Sciences, Lanzhou University, Lanzhou Gansu, China

Hamid Mahasin

Ya‐nan guo, de‐gui wang, associated data.

Not applicable.

Accurate and integral cellular DNA replication is modulated by multiple replication‐associated proteins, which is fundamental to preserve genome stability. Furthermore, replication proteins cooperate with multiple DNA damage factors to deal with replication stress through mechanisms beyond their role in replication. Cancer cells with chronic replication stress exhibit aberrant DNA replication and DNA damage response, providing an exploitable therapeutic target in tumors. Numerous evidence has indicated that posttranslational modifications (PTMs) of replication proteins present distinct functions in DNA replication and respond to replication stress. In addition, abundant replication proteins are involved in tumorigenesis and development, which act as diagnostic and prognostic biomarkers in some tumors, implying these proteins act as therapeutic targets in clinical. Replication‐target cancer therapy emerges as the times require. In this context, we outline the current investigation of the DNA replication mechanism, and simultaneously enumerate the aberrant expression of replication proteins as hallmark for various diseases, revealing their therapeutic potential for target therapy. Meanwhile, we also discuss current observations that the novel PTM of replication proteins in response to replication stress, which seems to be a promising strategy to eliminate diseases.

Accurate DNA replication is modulated by multiple replication‐associated proteins, which is fundamental to preserve genome stability. Abundant replication proteins are involved in tumorigenesis and development, implying these proteins act as therapeutic targets in clinical. Replication‐target cancer therapy emerges as the times require. Furthermore, the novel posttranslational modification of replication proteins in response to replication stress, which seems to be a promising strategy to eliminate diseases.

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1. INTRODUCTION

Accurate, faithful, and error‐free DNA replication is a vital prerequisite to ensure normal operation for the entire biological processes. DNA replication is an intricate and ingenious procedure that is fundamental to cellular life. Incomplete or erroneous DNA replication events lead to aberrated cell cycles, gene mutations, and gene copy number variations, further resulting in diseases, even cancer. 1 , 2

DNA replication can be roughly separated into three typical sections: (1) DNA replication initiation, in which the replication origins are prepared to unwind the DNA helix; (2) DNA replication elongation, in which replisomes move in opposite directions via semi‐conservative synthesis; (3) DNA replication termination, when converging replication forks meet and replisome disassembly. 3 , 4 Integrated DNA replication events are tightly regulated from bacteria to eukaryotic cells to allow correct genetic information transmission through cell division. In whole process of DNA replication, random mistakes are a source of genomic instability, causing heritable mutations that drive cancer evolution. 5

Owing to aberrant DNA replication and constitutive growth signaling, cancer cells may experience “replication stress,” a phenomenon that delays DNA synthesis and is a hallmark of cancer. 6 , 7 To safeguard precise duplication of the entire genome, cells initiate the DNA damage response (DDR) mechanisms to account for the continuous barriers. 8 The DNA repair pathways in mammalian cells accurately repair distinct types of DNA damage, whereas DNA repair dysfunction can predispose organisms to disease. Nevertheless, the DDR system may be defeated to maintain the genomic integrity due to oncogenes activation or tumor suppressor genes inactivation. Therefore, exacerbating DNA replication stress (RS) as well as targeting DNA repair defects in cancer cells is an effective strategy for treating cancer specifically. 9

Posttranslational modifications (PTMs) of proteins could affect their functions in positively or negatively way, impacting multiple biological processes such as DNA replication, gene transcription, and DDR. 10 , 11 Recent studies supported that PTMs of replication factors have an extraordinary effect on DNA replication and respond to RS. Thus, the advanced understanding of modification of replication licensing factors and their implications for DDR may provide a novel insight into the cancer therapeutic target.

In this review, we elaborate on the overall DNA replication mechanism and summarize the comprehensive approaches that are aiming harness RS to target cancer. Furthermore, we explore the latest strategies and novel ideas to improve the efficacy and specificity of anticancer therapies. Meanwhile, we also enumerate the multifarious PTMs, elaborating how PTMs of replication proteins mediated DNA replication, RS response, DNA damage repair, and oncogenesis mechanism, which may provide a polynary insight into tumorigenesis and tumor therapeutics.

2. THE BASICS OF EUKARYOTIC DNA REPLICATION

2.1. dna replication initiation.

Mini‐chromosome maintenance (MCM) proteins are composed of six subtypes, MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7. All subunits integrate into a hetero–hexameric complex and act as a replicative DNA helicase to unwind the parental DNA. 12 Beyond that, other MCM proteins, MCM8, MCM9, and MCM10, are reportedly essential for the DNA replication and remaining genome maintenance. 13 , 14 , 15

In eukaryotic cells, activation of replication origins is a prerequisite of DNA replication, manifesting the bidirectional movement. 16 , 17 The prereplication complex (pre‐RC) forms at the origin recognition complex (ORC1‐6), which serves as the actuated operator of DNA replication. In order to maintain genome integrity, ORC proteins are essential for establishing pre‐RC at origins since the distribution and density of origins have to be adequate to replicate the entire genome without leaving any regions un‐replicated. In the early G1 phase, cell division cycle 6 (CDC6) and DNA replication factor 1 (Cdt1) are recruited to the replication origins, subsequently attracting MCM2‐7 complex to load onto chromatin. 18 , 19 MCM2‐7 hexamer itself has restricted helicase activity, while executing integrated helicase activity in combination with Cdc45 and GINS (CMG) during G1/S transition. 20 , 21 Those proteins could compose of preinitiation complex (pre‐IC), which then preparing to form bi‐directional replication forks once MCM double hexamers separating into two single units. 22 Additional factor, MCM10, collaborates with polymerase ε (polε) and polymerase δ (polδ) in replication origins for replication initiation, meanwhile interacting with CMG helicase to stabilize the replisome. 23 , 24 Moreover, one recent study found that MCM10 is necessary for CMG to transit between double‐strand DNA (dsDNA) and single‐strand DNA (ssDNA). Additionally, MCM10 migrates along with the replication fork and energizes replication elongation (Figure 1A ).

An external file that holds a picture, illustration, etc.
Object name is MCO2-4-e210-g005.jpg

The schematic diagram for DNA replication. (A) DNA replication initiation procedures are described in the text. In the early G1 phase, cell division cycle 6 (CDC6) and DNA replication factor 1 (Cdt1) are recruited to replication origins, subsequently cooperating with MCM2‐7 to load onto chromatin. MCM2‐7 complex interacts with GINS and Cdc45 as CMG complex to initiate DNA replication. (B) DNA replication elongation. CMG helicase initiates double‐strand DNA unwinding. Two replicative polymerases, polε and polδ, rely on PCNA to principally execute DNA synthesis to elongate the nascent leading and lagging strands, respectively. Additionally, flap endonuclease 1 (FEN1) and topoisomerases (TopoI, TopoII) safeguard typical and efficient DNA polymerization. (C) DNA replication termination. CMG removal from the strand represents replication termination. CDC48/p97‐mediated MCM7 polyubiquitination and CDK‐mediated ORC phosphorylation facilitate CMG disassembly, thus leading to CMG unloading.

In light of the bulky size of genomes and abundant amounts of chromosomes, eukaryotic cells contain numerous replication origins to duplicate their genomes. Nonetheless, massive replication origins assist high‐efficiency genetic information transfer with more hazard since their distribution and proceeding have to be tightly controlled. Eukaryotic cells have an extensive system to guarantee precise DNA replication. During the S phase, disparate genome regions or domains are duplicated at a staggered time, and the origin licensing system is carried out from firing at distinct cell cycle phases. In addition, in the G1 phase, license origins are much more than they used in the subsequent S phase, while the inactive origins are named as “dormant origins.” The plain fact is that dormant origins constitute the tremendous majority of licensed origins, which serve as backup to sustain the replication fork regular progression under conditions of RS.

Since overabundance and distribution patterns on chromatin, the definite and accessional functions of MCM proteins are always contradictory. The issue is defined as “MCM paradox,” which is chiefly embodied in two aspects: (1) MCM2‐7 complexes massively exist in nonreplicated DNA; (2) excess MCM hetero–hexamers attach to chromatin instead of replication origins and ORCs. 25 , 26 Apparently, the excess MCMs are involved in other biological processes. Numerous studies proved that MCMs serve as biomarkers in multiple tumors, which are closely related to tumorigenesis, development, and even in tumor therapeutic.

2.2. DNA replication elongation

Afterward, dozens of distinct proteins consistently coordinate to promote DNA replication elongation. Owing to the DNA antiparallel structure and DNA polymerases’ 3′–5′ direction of forward motion, the running replication forks separate into two single strands, which are continuously synthesized leading strand and inconsecutive synthesized lagging strand, respectively. 27 In the lagging strand, discontinuous and short fragments are considered as Okazaki fragments, which require DNA ligase to assemble into the complete lagging strand rapidly and ultimately. 28

DNA elongation and polymerization is catalyzed by multifarious enzymes, which are responsible for DNA synthesis and progression of the DNA replication. Polymerase α (polα)/primase mainly partakes in the initial stage of DNA synthesis. 29 Four subunit enzymes of polα/primase catalyze RNA oligonucleotide synthesis, which subsequently can be applied to extend by a short stretch of DNA. After this initiation step, polα is immediately switched into replicative polymerase via an ATP‐dependent manner. 30 Two replicative polymerases, polε and polδ, principally execute DNA synthesis to elongate the nascent leading strand and lagging strand, respectively. Both polε and polδ are four subunit enzymes with intrinsic 3′–5′exonuclease proofreading activities, which increase replication fidelity with a lower mutation rate. 31 , 32 Moreover, multiple evidence suggested that polymerase activities of polδ are stimulated by protein proliferating cell nuclear antigen (PCNA), serving as a platform to coordinate numerous proteins interaction at the replication fork. Polδ cooperates with PCNA to promote long stretches of DNA synthesis. 33 Nevertheless, PNCA could not load onto DNA without replication factor C (RFC) assistance, which could wrap PCNA homo‐trimeric ring to promote its DNA loading via ATP‐dependent manner. 34 Additionally, flap endonuclease1 (FEN1) and Dna2, two endonucleases, are mainly needed for DNA and RNA flap structure cleaving, which are mediated by replication protein A (RPA). 35 Ultimately, flap cleavage generated DNA nick is sealed by DNA ligase I (Figure 1B ). 36

2.3. DNA replication termination

In contrast to the initiation and elongation steps, DNA replication termination still remains several queries, even though it occurs on neighboring replication origins encounter. Due to the torsional strain caused by DNA helicase, positive supercoils structure must be removed by DNA topoisomerases to maintain the replication fork progression and genomic integrity. Type I and type II topoisomerases unwrap supercoils primarily to rotate the direction of fork evolution into clockwise, which could transfer the topological stress. Additionally, type II topoisomerase specifically removes precatenanes to assure converging replisomes unwind and DNA complete synthesis. 37 , 38

Like the ultraprecise instrument, every module of DNA replication all links with one another. During the S phase, reloading of MCMs is inhibited to ensure that no genome segment is re‐replicated to preserve genome integrity. 39 Except for MCM proteins, several proteins are involved in this node. Cdt1, as the component of pre‐RC, is the prime modulator to prevent re‐replication. 40 CDK‐mediated phosphorylation of Cdt1 is inhibited from interacting with Orc6 once the DNA replication initiation in Saccharomyces cerevisiae . 41 In eukaryotes, however, Cdt1 is degraded upon S phase entry through two independent ubiquitin‐mediated pathways. 42 , 43 In addition, other components of pre‐RC, ORC1‐6, are also the critical point for preventing re‐replication. 44 During S phase entry, ORC1 is released from replicating sequence via CDK‐mediated manner, which prevents ORC from entering second round of licensing. 45 CDC6 also manifests in preventing re‐replication via a distinct mechanism. Some studies support that the SCF cyclin F ubiquitin ligase complex impedes DNA re‐replication by proteasomal degradation of CDC6 in the cell cycle. 46

In eukaryotes, neighboring CMG complexes meet each other on different strands, which is propitious to stable and orderly replication progression. Leading and lagging strand separation promotes CMG of one replisome to straightway transfer into the lagging strand template. Rapid encounter of adjacent CMG complex without pausing could preserve genome stability, whereas suspending for a while when CMG complex confronts a covalent DNA–protein. 47 This observation points out that cells set the defense mechanism to prevent conflict between adjacent CMG complexes during DNA replication termination.

CMG removal from the strand is a remarkable event in eukaryotic replication termination when CMG cooperated with dsDNA. Previous reports suggested that converging CMG complexes proceed migration along the leading strand template until the downstream Okazaki fragment, which no longer performs dsDNA unwinding at all. Ultimately, CRL2 Lrr1 ‐mediated MCM7 polyubiquitination leads to CMG unloading, subsequently removed by CDC48/p97 segregase (Figure 1C ). 48

DNA replication is an intricate process with a coordinated interplay of multiple proteins. As we summarized, each step of DNA replication must be strictly regulated to preserve genome integrity, while internal or external DNA‐damage agent always threatens DNA replication to activate DDR system. Meanwhile, dysfunction of DNA replication and DDR causes severe diseases, which highlights the role of DNA replication in tumorigenesis and development.

3. EVOLUTION OF THE CORE REPLICATION PROTEINS

3.1. cmg complex.

In eukaryotes, DNA replicative helicase CMG complex binds to dsDNA at replication origins, subsequently transfers to ssDNA for DNA unwinding. As we described above, Cdc45 and GINS cooperate with MCM2‐7 during S phase entry, forming CMG helicase for bidirectional replication forks (Figure 2A ). 49

An external file that holds a picture, illustration, etc.
Object name is MCO2-4-e210-g002.jpg

General crystal structure of CMG complex and PCNA. (A) Crystal structure of the CMG complex. The single‐strand DNA (ssDNA) is colored lavender and each CMG units are uniquely colored and labeled. (Pictures from Protein Data Base mark as 6XTX. 3D PFV: 6XTX (rcsb.org).) (B) Crystal structure of the MCM2‐7 complex. MCM units are uniquely colored and labeled. (Pictures from Protein Data Base marks as 3J48. RCSB PDB ‐ 3JA8: Cryo‐EM structure of the MCM2‐7 double hexamer.) (C) Crystal structure of GINS. GINS units are uniquely colored and labeled. (Pictures from Protein Data Base mark as 2Q9Q.RCSB PDB ‐ 2Q9Q: The crystal structure of full‐length human GINS complex.) (D) Crystal structure of PCNA as viewed from top and side. Three subunits are uniquely colored and labeled. (Pictures from Protein Data Base mark as 3JA9. RCSB PDB ‐ 3JA9: Structure of native human PCNA.)

MCM proteins were firstly identified in S. cerevisiae , which were deemed to MCM. Based on electron microscopy investigations, each MCM monomer involves two conserved main domains exercising respective functions. 50 , 51 MCM2‐7 complexes could motion via the nuclear localization signals on N‐terminal region of MCM2 and the C‐terminal of MCM3, whereas nuclear export signals on the central part of MCM3. 52 In contrast, recent data suggest that MCM complexes are shuttled in interphase cells basically relying on the nuclear export signals in MCM3. 53

The N‐terminal domain (MCM N ) possesses three consistent crystal structure subdomains: an OB (oligonucleotide/oligosaccharide binding)‐fold, a peripheral helical bundle, and usually a zinc‐binding motif (Figure 2B ). 54 , 55

The OB‐fold subdomain links two single hexamers as head‐to‐head form and is also for DNA binding. 51 Mcm4 Chao3 (chromosome aberrations occurring spontaneously 3) mutation occurring in mouse Mcm4 OB fraction disrupts routine DNA binding process, resulting in genomic instability. 56 , 57

The peripheral helical bundle interacts with the OB fraction via a slight linker promoting the interaction with DNA. 58 The observation indicated that a helical bundle might be essential for protein–protein interplay and protein–DNA interactions during the initiation step. However, molecular mechanics studies presented that deleting a helical bundle exerts a limited influence on MCM function. 59 , 60

The X‐ray crystal structures of MCM N suggested a zinc‐binding domain, which presented two conserved arginine residues in Pyrococcus furiosus MCM ( pf MCM). Studies with the pf MCM verified that the zinc‐binding domain is probably needed for ssDNA binding. 54 Mutation of these two conserved arginine residues in MCM4/6//7 interfered with the loading of MCM2‐7 complex onto DNA, further resulting in growth defect in S. cerevisiae . These findings suggested that zinc‐binding domain of MCM4/6/7 is the vital region in ssDNA binding and origin melting. 61 , 62 In eukaryotes, the zinc‐binding motif of MCM3 lacking a prominent motif impacts the MCM2‐7 complex original function, suggesting that zinc‐binding motifs play a vital role in MCM2‐7 activities. 63

MCM proteins contain an AAA + ATPase domain in C‐terminal with two subunits terming as Walker A and Walker B, which are integrant for ATP hydrolysis and ATP binding. 64 , 65 Mutation of nearly any residues of the MCM AAA + ATPase domain eradicates ATPase activity. 66 Despite all the MCMs harboring ATP‐binding motifs at the intersubunit interfaces, the ATP‐binding mode is quite different. 67 MCM4/6/7 proteins exhibit distinct functions when the ATP binding sites undergo mutations. 68 It should also be mentioned that MCM7 is required for ATP hydrolysis and DNA helicase via its ATP binding motif. 69

Nuclear magnetic resonance (NMR) structure studies revealed that the C‐terminus of MCMs comprises a winged helix (WH) domain. Furthermore, the WH domain connecting to the AAA + ATPase domain exhibits ATPase activity, promoting the domain shift via a flexible linker with the protein core. 70 , 71 In contrast, archaeal MCM exhibits increasing ATPase activity and dsDNA unwinding activity when partial deleting of WH domain. 72 Thus, WH domain may reserve the latent function during dsDNA unwinding and may take effect in initiating helicase activity. 73

Except for conserved MCM2 and MCM3, MCM8 and MCM9 also possess a nuclear localization signal to shuttle between cytoplasm and nuclear. 74 Some studies indicated that MCM2 and MCM3 distribute in the cytoplasm but temporally and spatially shift to nucleus in a cell cycle‐dependent manner. 52 However, the distribution of MCM2 may also be associated with DNA damage. Envelope protein gp70 directly recognized MCM2 nuclear localization signal in the cytoplasm, thus enhancing DNA damage‐induced apoptosis. 75 , 76 However, limited researches discuss the purpose of the MCM proteins motion.

The eukaryotic GINS complex consists of four subunits, Sld5, Psf1, Psf2, and Psf3, pronounced as Sld‐ g o, Psf‐ i chi, Psf‐ n i, and Psf‐ s an in Japanese. Despite its central role in CMG complex, GINS also modulates massive protein interaction during DNA replication and DNA repair. 77 Each subunit of GINS interacts with each other extensively, meanwhile, each of them possesses the related two‐domain (A‐domain: α‐helical region; B‐domain: β‐rich region) structure, whose structural similarity causes pseudo‐twofold symmetry in whole GINS architecture (Figure 2C ). 78

In eukaryotic GINS, Psf1 only has an intact A‐domain, yet B‐domain is invisible in the crystal lattice, even though the similar B‐domain of Psf1 to the three other subunits via sequence alignment. Some reports indicated that the complementarity of the B‐domain into Psf1 disturbs GINS packing, which implies an essential role in CMG formation and Cdc45 binding. 79 However, the Psf3 B‐domain is widely considered to interact with the MCM complex, strengthening the MCM3–MCM5 interface. 78

In the CMG complex, Cdc45 cooperates with the MCM2‐7 complex to shut down the MCM2–MCM5 gate, which is crucial for ATPase site forming and CMG translocation on ssDNA. Cdc45 possesses a distinct helical motif, which is proximal to the catalytically active domain of polε. N‐terminus of polε crosslinks with Cdc45 on the tip of the protrusive helix of Cdc45, indicating Cdc45 impacts on CMG helicase and polε polymerase activity. 80

3.2. PCNA and its binding proteins/enzymes

Eukaryotic sliding clamp protein, PCNA, is a ring‐shaped homo‐trimer with each subunit containing two domains, which presents a pseudo‐six‐fold symmetry pattern. Each subunit of eukaryotic PCNA is formed from two independent and semblable folded domains, which is ultimately confirmed by X‐ray crystal structure analysis. PCNA could be roughly separated into two domains, domain A and domain B, connected by an extended β sheet across the interdomain frontier. Moreover, a flexible linker concatenates two domains are named the interdomain connector loop. The assembled pattern among three subunits performs end to end structure, precisely as one domain A connects with the adjacent subunit's domain B (Figure 2D ). 81 , 82

Due to its essential role in DNA replication, PCNA embraces the DNA and travels along it, conducting for DNA polymerases and DNA replication proteins. DNA could cooperate with three equivalent sites of PCNA since its symmetry patter. PCNA sliding along DNA counts on its basic residue interactions with the phosphates of DNA, which promoting the rotation of PCNA around the DNA. One convincing model supports that PTMs of PCNA alter its positive charges on the inner side, leading to unconscionable movement. Thermal and chemical denaturation researches demonstrated that human PCNA is much more unstable than S. cerevisiae homolog though they share the homogeneous three‐dimensional structure. Furthermore, human PCNA performs tough backbone dynamics, especially at helix of ring inner surface. Due to the highly dynamic and plastic property, PCNA evolves as platform to facilitate interacting with multiple proteins. 83 , 84

A huge collaborative network of proteins engages for high fidelity DNA repair and accurate DNA damage repair. PCNA is regarded as entire hub in DNA replication that interacts with abundant proteins involved in multiple DNA‐related processes. By occasion of homo‐trimer shape of PCNA, three identical pockets could cooperate distinct partners simultaneously and coordinate various functions spatiotemporally. Numerous PCNA‐interacting proteins (PIP) interact with PCNA via their PIP box. A typical consensus amino acid sequence of PIP motif is (Q‐x‐x‐(I/L/M)‐x‐x‐(F/Y)‐(F/Y)). 83

The PCNA ring has three independent PIP‐box binding sites with three distinct ligands for binding proteins. To secure normal replication, three promoters of the PCNA trimer convene DNA ligase I, polδ, and FEN1 simultaneously to ensure stable Okazaki fragment synthesis. Constitutive complex has been demonstrated in yeast called the “PCNA tool belt,” which could be modulated by diverse PTMs. FEN1 interacts with PCNA via its canonical PIP box exhibiting lower affinity, while increasing the affinity by replenishing 20‐residue long PIP fragments. 81 These observations indicate that PIP box of proteins mediates their interaction affinity to PCNA, which is also modulated by PTMs. Thus, targeting such a binding site may interfere with DNA replication and DNA damage repair, thereby serving as attractive targets for cancer therapy.

Indispensable DNA replicative polymerases are required for DNA synthesis with high efficiency and accuracy. The general architectures of DNA polymerases present right‐hand aspect with three main functional domains, which also contain exonuclease activity site for proofreading. Eukaryotic DNA replication primarily depends on three B‐family DNA polymerases: polα, polδ, and polε. Polε and polδ are chiefly for high accurate DNA synthesis on the leading and lagging stands via interaction with PCNA, respectively. All eukaryotic replicative polymerases contain two conserved motifs with cysteines (CysA and CysB), which was regard as Zn‐finger motifs originally. Except for detectable PIP box sequence in polδ, CysA motif could also directly interact with PCNA to promote efficient loading and synthesis of DNA. 31 However, little is known about how pol ε with PCNA in mammals.

Insight into the general architecture of the replication proteins assists us in clarifying more accurate molecular regulatory mechanisms. Due to the complex interaction network, spontaneous or revulsive mutations of MCM2‐7 complex disturb the normal biological processes such as DNA replication, cell proliferation, and DDR. 57 , 85 Moreover, MCMs load onto DNA via particular binding domains, thus it is possible to interrupt the chromosome remodeling through interfering these specific domains. 26 , 86 PTMs of replication proteins in diverse residues distributed in different domains may present a special effect due to their topological alteration in positive or negative patterns. Nevertheless, the precise regulatory for how PTMs in different domain affecting downstream processes is still unclear.

4. THE REPLICATION STRESS RESPONSE

DNA is constantly threatened by various DNA damage stimulus including ultraviolet (UV) light, ionizing radiation (IR), biochemical reagent, which disrupting normal DNA replication and leading to RS. 87 , 88 The RS leads to replication fork stalling and even collapsing if the stress cannot be solved immediately. 89 RS‐induced mitotic abnormalities can activate DNA damage repair pathway and result in activation of oncogenes. 88 , 90 Mutually, activation of oncogenes aggravates RS and genomic instability in human cancer cells. 91

If the RS cannot be fixed immediately, the replication fork will collapse thus causing DNA strand breaks. 92 To ensure ordinary cellular events against stalled replication forks, cells harbor multiple DDR pathways to preserve genomic integrity. 93 DNA repair pathway fixing damage sites is subject to the particular DNA damage types. In general, nucleotide excision repair (NER) is required to fix the UV light‐induced single‐strand breaks (SSBs) and bulky lesions. 94 Abnormal DNA bases‐ and oxidative damage‐induced intermediates are commonly repaid by base‐excision repair (BER), whereas correct insertion loops are repaired by mismatch repair (MMR). 95 , 96 The most lethal and fearful damage type is IR‐ or chemically induced double‐strand breaks (DSBs). Classic pathways to repair DSBs are homologous recombination (HR) and nonhomologous end‐joining (NHEJ). 97 , 98 In addition, cell cycle checkpoint activation is also regarded as a vital DDR pathway, which includes Rad3‐related serine/threonine kinase (ATR)‐checkpoint kinase 1 (CHK1) and the ataxia telangiectasia‐mutated serine/threonine kinase (ATM)‐checkpoint kinase 2 (CHK2) pathway (Figure 3 ). 99

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DNA damage response framework. DNA is constantly threatened by various DNA damage stimulus including ultraviolet (UV) light, ionizing radiation (IR), biochemical reagent, which disrupting normal DNA replication and leading to single‐strand break (SSB), double‐strand break (DSB), replication stress (RS), and base mismatch. DNA damage triggers sequential cascade reactions promoting cellular survival, including DNA damage repair and cell cycle checkpoint activation. Severe DNA damage may ultimately result in cell death via apoptosis. DNA repair pathway fixing damage sites is subject to the particular DNA damage types. Nucleotide excision repair (NER) is required to fix the UV light‐induced SSBs and bulky lesions. Abnormal DNA bases‐ and oxidative damage‐induced intermediates are commonly repaid by base‐excision repair (BER), whereas correct insertion loops are repaired by mismatch repair (MMR). Classic pathways to repair DSBs are homologous recombination (HR) including single‐strand annealing (SSA) and non‐homologous end‐joining (NHEJ).

Genomic instability is the hallmark for cancers, which is related with massive unsolved DNA damage. Based on the characteristics of cancer cells, DNA‐damaging chemotherapy is widely applied clinically even though accompanied by severe side effects to normal tissues. Given the elementary function of the DDR, DDR‐target therapy has a putative role to intercept cancer cells’ rational response through combination treatment to patients lacking specific DDR functions. Apparently, probing into the mechanisms of DNA damage repair in cancers might be an absorbing strategy for cancer therapeutic target. Since interrelated relationship between DNA replication and DDR, multiple crucial DNA replication factors are involved in DDR including MCM proteins, CMG complex, and PCNA. Intriguingly, multifunctional roles of these proteins are optimal target for cancer treatment.

RS blocks the routine DNA replication and sticks normal cell cycle, activating the cell cycle checkpoint mechanism. 100 Since stalled replication fork forms the exposed ssDNA, RPA primarily recognizes naked ssDNA to protect it against breakage. Numerous evidences revealed that RPA serves as the most frequently responsive protein after DNA damage or during DNA repair. RPA‐coated ssDNA then unites to recruit ATR via its partner protein ATRIP (ATR‐interacting protein). 101 Subsequently, ATR activation elicits cell cycle checkpoints and stabilizes the replication fork via phosphorylating its downstream effector kinase CHK1, further preventing damaged DNA from entering mitosis. ATR activity is also stimulated by DNA topoisomerase 2‐binding protein 1 (TopBP1), promoting its role in phosphorylating the substrates. 102 Numerous studies indicated that the ATR‐CHK1 pathway mainly prevents S phase progression and further mediates DNA damage repair. 103 , 104 The function of ATR may be interrupted by numerous factors such as MCM7. 105 Partial depletion of MCM7 directly leads to UV‐induced ATR activation defect. 106 C17orf53 is one of the uncharacterized genes involved in ATR response. 107 Some studies characterize that C17orf53 protein might interact with RPA1 and MCM8‐9 to regulate DNA replication and respond to DNA damage. 108 The collapsed replication fork generates DSBs, which stimulating the DDR processes, indicating a tight relationship between DDR and DNA replication. 109

As the MCM paradox query, abundant amounts of MCM2‐7 are exciting in most growing cells, whereas only a tiny proportion of these are used for DNA replication. Several striking outcomes have been revealed that redundant MCM proteins may serve as “backups” to ensure adequate dormant replication origins activating when suffering RS, such as in the presence of aphidicolin. 110 , 111 Furthermore, knockdown of MCM2‐7 increases the frequency of chromosome breaks, thus causing cells hypersensitive to RS in eukaryotes. 112 In Drosophila , depleting MCM2 does not affect cell growth rate, whereas partial reduction of MCM2 decreases the number of spendable origins. 113 In contrast, knockout of MCM7 activates checkpoint signaling in human cancer cells, prohibiting their unbitted DNA replication, which may act as the potential target for cancer treatment. 114 Some reports also support that partial depletion of MCM2‐7 in HeLa cells does not show any noticeable impact on cell viability, whereas resulting in lethally hypersensitive to hydroxyurea (HU). 112 Meanwhile, deletion of MCM5 also could not effect cell proliferation but makes cervical cancer cells vulnerable to RS such as HU or aphidicolin. 115 These findings prove that excess MCM2‐7 proteins safeguard the cells against replicative stress by licensing dormant origins.

Bai et al. 116 demonstrated that chronic RS lessened MCM2‐7 expression via a p53‐mediated manner. During exposure to low‐level RS, MCM proteins are gently decreased accordance with RNAi‐related gene silencing. The microRNA (miRNA)‐34 family targets MCM5 directly, causing descending expression of other MCM proteins and negatively regulating cell cycle progression when overexpression of these miRNAs. 116 The eukaryotic whole‐genome analysis investigated MCM4 N‐terminal serine/threonine‐rich domain (NSD) segments combined with Rad53, Sld3, and Ddf4, to activate origin and promote replication progression to respond to RS. 117 , 118

The ATM and CHK2 kinases are critical regulators of double‐strand DDR. ATM activation requires the MRN (Mre11–Rad50–Nbs1) DSBs sensor complex that processes DNA ends and ATM to broken DNA molecules. 119

The Bloom syndrome DNA helicase (BLM) is part of HR to maintain chromosome stability and promotes DNA replication after repair of DNA damage. 120 , 121 Shastri et al. 122 identified BLM helicase interacts with MCM6 to resist HU‐induced RS just in S‐phase and keeps the routine DNA replication. In contrast, BLM–MCM6 is needed for cell survival under pyridostatin (RR82) induction in S‐phase, suggesting the BLM–MCM6 complex partakes in DNA replication and responds to DNA damage in eukaryotes. 122 , 123 Since phosphorylation of BLM shows an ATM‐dependent manner, it provides us with a possible that BLM–MCM6 complex may be regulated by ATM in DNA damage repair. Moreover, Fanconi anemia (FA) complementation group D2 (FANCD2) can directly connect to MCM2‐7 complex upon RS, thereby preventing pathological replication structure's accumulation 124 , 125 Naturally, FANCD2 has closely relationship with ATM, indicating ATM indirectly modulates the MCM proteins in answer to DNA RS and DNA damage.

Substantive results revealed that MCM8 and MCM9 play a vital role in HR repair as MCM8‐9 complex. 126 Lee et al. 127 found that incapable mutation of MCM8‐9 complex could not recognize MRN complex, leading to degressive HR efficiency. Moreover, some research proved that the depletion of MCM9 is attributed to reduced proliferation, which may be modulated by ATM‐CHK2 pathway. The MCM‐binding protein (MCMBP) is considered as a chaperone of MCM proteins to assist dynamic assembly of the MCM2‐7 hexamer and promotes MCM8‐9 for HR repair. MCM proteins connecting with MCMBP is essential for maintaining the pool of functional MCM2‐7 hexamers. 128 , 129

MCM10, components of the replication fork, loads to DNA after MCM2‐7 complex settling down. MCM10 associates with Dna2 may function on the lagging strand during DNA replication, while Dna2 physically interacts with ATM at DNA damage sites. These complicated molecular connections imply the MCM10 potential function in stalled replication fork and DNA damage area. 130 , 131 One possible explanation for this circumstance is that the MRN complex stabilizes replisomes at stalled forks and recruits multiple factors to fix the predicament. 132 Therefore, MCM10 cooperating with DSB repair proteins could exhibit one direct role of MCM10 in mediating DSB repair (Figure 4 ).

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MCM proteins in response to DNA damage. RPA recognizes DNA damage‐induced single‐strand DNA (ssDNA) forming RPA‐coated ssDNA. RPA‐coated ssDNA recruits ATR via ATR‐interacting protein (ATRIP). ATR interacts with MCM7 and TopBP1 to activate CHK1 phosphorylation. Activation of ATR‐CHK1 could further lead to DNA repair activation. MCM proteins act as an intermediary in DNA repair and DNA checkpoint reactions. MRE11–RAD50–NBS1 (MRN) complex recognizes double‐standard break sites (DSBs) and recruits γ‐H2AX to DSBs. MCM proteins, especially MCM8–MCM9, are recruited by the MRN complex and cooperate with ATM to activate CHK2 phosphorylation leading to DSBs repairs such as HR and NHEJ. Multiple DNA repair proteins are recruited to the damage sites to perform distinct repair pathways, such as XPC, BRCA1, 53BP1, and so on.

In conclusion, abundant MCM proteins act as a reserve to safeguard the DNA replication under DNA RS, nay, interact with multiple DNA damage factors to perform the DNA damage repair via ATR–CHK1 and ATM–CHK2 pathways. More stirring, numerous studies revealed that MCMs are phosphorylation substrates of ATM and ATR. Thus, it is remarkable to clarify how the PTMs of MCMs modulate the DNA damage repair pathway.

5. PTMS OF PROTEINS TARGET DNA REPLICATION AND DNA DAMAGE IN DISEASE

It is conceivable that the replication proteins’ dynamic status is regulated by known kinases such as ATM and ATR, whereas these proteins also undergo additional regulatory mechanisms. Substantive publications have revealed distinct replication proteins exercise unpredictable functions achieved by diverse PTMs. 11 Such PTMs contain phosphorylation, ubiquitination (Ub), small ubiquitin‐like modifier (SUMOylation), O‐N‐acetyl‐ d ‐glucosamine (GlcNAcylation), and acetylation. Recent results revealed that PTMs of these proteins contribute to DNA replication and DNA damage repair, which also could be potential therapeutic target for tumors. 133

5.1. MCMs phosphorylation

Individual MCMs are subject to phosphorylation in a cell cycle‐specific manner, which may be consistent with their cell cycle‐specific functions. Due to various kinase types, phosphorylation of MCM proteins undergoes distinct regulatory mechanisms. Moreover, exceptional phosphorylation of MCMs might disrupt DNA replication progression, further causing DNA damage and leading to diseases or cancers (Table 1 ). 134

Summary of the MCMs modification in response to DNA replication and DNA damage

Modification type	Mediator	Substrate	Function	Reference
Phosphorylation	CDK	MCM2‐S139	Promote chromatin loading
		MCM3‐S112	Promote MCM2‐7 incorporation
		MCM3‐S711	Cell cycle regulation
		MCM3‐T722	Promote chromatin loading
Phosphorylation	CDK	MCM4‐T7, T9, S32, T110	Decrease the binding of MCM to DNA
		MCM4‐S3, S32	Activation cell cycle checkpoint	,
		MCM7‐S121	Promote chromatin loading	,
		MCM7‐S365	Cell cycle regulation	,
	DDK	MCM2	Interact with CDC45 and GINS
		MCM2‐S27, S41, S139	Maintain genome integrity
		MCM2‐S164, S170	Proper response to DNA damage	,
		MCM4‐NSD	Cell growth and S phase progression	,
		MCM6‐NSD	Cell proliferation	, ,
		MCM10	Facilitate double hexamer separation	,
	ATR	MCM2‐S108	Response to DNA damage
		MCM6‐S13	Response to DNA damage
	ATM	MCM3‐S535	Cell cycle checkpoints activation
		MCM3‐S725, S732	Response to DNA damage
	ATR/ATM	MCM10	Response to DNA damage
Ubiquitination	KEAP1	MCM3	Preserve genome stability
	HERC2	MCM6	Preserve genome stability and DNA repair	, ,
	UBE3A	MCM6	Preserve genome stability and DNA repair	, ,
	CDC49/p97	MCM7	ICL repair	,
	BRCA1	MCM7	ICL repair and HR	,
	SCF	MCM2‐7	CMG helicase disassembly
	CUL	MCM7	Preserve genome stability	,
	TRAIP	MCM7	CMG helicase disassembly, ICL repair	, ,
	CUL4	MCM10	Preserve genome stability	,
SUMOylation	Slx5/Slx8	MCM2‐7	In response to replication stress	,
	Mms21	MCM2, MCM3	Preserve genome stability	, ,
	Ulp1/Ulp2	MCM4, MCM7	Preserve genome stability
	Siz1, Siz2	MCM2, MCM3, MCM4, MCM5, MCM7	Preserve genome stability
		MCM2, MCM3, MCM4, MCM7	In response to cytotoxic stress	, ,
		MCM10	Preserve genome stability
Acetylation	HBO1	MCM2	Preserve genome stability	, ,
	MCMAP	MCM3	DNA replication
	p300	MCM10	Stabilize genome integrity
	SIRT1	MCM10	Stabilize genome integrity
O‐GlcNacylation	OGT	MCM2‐7	Preserve genome stability
Methylation	KMTS	MCM2‐7	Response to heat stress

5.1.1. CDK/DDK‐mediated phosphorylation

Cyclin‐dependent kinases (CDKs) and their regulatory proteins cyclin are main protein kinases to modulate the progression from G1 into S phase and from G2 into mitosis. Thus, different CDKs–cyclin assemblages phosphorylate MCMs to influence the cell cycle progression or DNA damage repair pathway.

MCM2 and MCM3 are generally phosphorylated by CDK2 at Ser‐139 and Ser‐711 in eukaryote, respectively. 135 MCM3 phosphorylation at Thr‐722 promotes MCM complex chromatin loading, which is medicated by cyclin E/CDK2. 136 What is more, cyclin E/CDK2‐mediated MCM7 phosphorylation at Ser‐121 in HL‐7702 cells also facilitates its chromatin loading and normal mitosis. 137

MCM3 Ser‐112 is phosphorylated by CDK1, promoting the connection among MCM subunits and MCM3 chromatin loading in U20S cells. 138 Alternatively, ineffective MCM3 phosphorylation may impair MCM2‐7 helicase activity, resulting in S phase delay and activating S phase checkpoint, ultimately causing a turbulent cell cycle. When stalled replication fork activated cell cycle checkpoint, abundant MCM proteins, especially MCM3 and MCM7, are assembled at damage sites to block the S phase entry. Uniformly, some research demonstrated that overexpression of the wild‐type MCM7 resulted in S phase block. MCM7 Ser‐121 is strongly phosphorylated by cyclin B/CDK1, whereas Ser‐365 is phosphorylated by CDK2. 137 , 139 These findings indicate that phosphorylation of MCM7 interferes its DNA loading ability. In contrast, dephosphorylation of MCM7 protects the cell cycle when confronting RS. Additionally, CDK1 phosphorylates MCM4 at Thr‐7, Thr‐19, Ser‐32, Ser‐88, and The‐110, while Ser‐3 and Ser‐32 are phosphorylated by CDK2. These modifications decrease the ability of MCM2‐7 to load onto DNA, avoiding re‐replication during mitosis. 140 More obviously, MCM4 is phosphorylated by CDK under HU and UV irradiation, which is critical to stimulate cell cycle checkpoint activation. 141 , 142

In addition, another DNA replication‐associated kinase, Dbf4‐dependent kinase (DDK), also is essential for the phosphorylation of the MCM2‐7 complex. DDK‐mediated phosphorylation of MCMs induces a conformational change, therefore impacting the connection with other DNA replication factors. The observation indicated that DDK‐dependent MCM2 phosphorylation dissociates from MCM5, unfolding the MCM2‐7 hexameric to prevent DNA re‐replication. Electron microscopy analysis revealed that the interaction of MCM2‐7 with CDC45 and GINS promote MCM2–MCM5 gap blocking. 143

Tsuji et al. identified three DDK‐dependent MCM2 phosphorylation sites (Ser‐27/41/139), both in vivo and in vitro. Deactivation mutation of MCM2 (Ser27/41/139‐Ala27/41/139) blocks DNA replication and causes RS, which suggests that DDK‐mediated phosphorylation of MCM2 closely regulates DNA replication. 144 In addition, other studies revealed that phosphorylation of MCM2 by DDK is critical for MCM2‐7 ATPase activity in vitro. Previous studies showed that phosphorylation of S. cerevisiae MCM2 by DDK at Ser‐164 and Ser‐170 is crucial for a proper response to DNA damage. 145 Further research demonstrated that the phospho‐deficient mutation of MCM2 (Ser164‐Ala, Ser170‐Ala) increased sensitivity to HU and base analog 5‐fluorouracil (5‐FU) as spontaneous mutation rate, which expressly revealed DDK‐mediated MCM2 phosphorylation modulated MCM2‐7 activity and preserved genome stability in response to replicative stress. 146 On the other hand, other research pointed to the NSD of MCM4 is the target DDK to promote S phase progression. 147 Taken together, DDK‐mediated phosphorylation of MCM2 and MCM4 serves as a critical point in modulating the MCM2‐7 complex dynamic motion and protecting the genome integrity.

Except for MCM2 and MCM4, MCM6 has an unstructured N‐terminal domain containing certain DDK target sites, and is phosphorylated by DDK in vitro. 148 Importantly, MCM4 and MCM6 NSD are phosphorylation in G1, S, and G2/M phase, which are vital for cell viability. Notably, inhibition of the MCM4/6 phosphorylation leads to additional growth defects, further causing genome instability. 149 Previous research demonstrated that DDK associated with MCM10 in vitro, which is consistent with an earlier finding in Schizosaccharomyces pombe . 150 MCM10 also interworks with MCM2‐7 to facilitate double hexamer separation, which is influenced by CDK and DDK‐mediated phosphorylation. 23

5.1.2. ATM/ATR‐mediated phosphorylation

According to the above description, MCM proteins are involved in the ATM/ATR signaling pathways to perform their DNA damage repair functions. In addition, ATM and ATR also serve as the master kinase to phosphorylate MCMs, stabilizing the DNA replication fork and actives cell cycle checkpoints. Cortez et al. 151 found that ATM phosphorylates MCM3 Ser‐535 under IR, whereas multiple DNA damage agents could cause ATR‐dependent MCM2 phosphorylation, such as radiation exposure and chemical reagents. Some reports also revealed that ATR‐mediated MCM2 is phosphorylated without stimulating DNA damage. 135 Further, ATM contributes to MCM3 C‐terminal Ser‐725 and Ser‐732 phosphorylation upon unstable condition. However, this phosphorylation may not cause MCM2‐7 complex conformational change. 152 Wagner et al. 153 found that MCM6 Ser‐13 was a novel putative ATR target site in answer to RS. UV irradiation disturbs DNA replication progression since MCM10 proteolysis in human cells. UV‐induced MCM10 degradation might be rescued by interfering with ATR/ATM inhibitor and CHK1 inhibitor, indicating that ATR and CHK1 kinase modulate its downregulation. 154 Taken together, ATR/ATM‐mediated MCMs phosphorylation is crucial for responding to DNA RS and DNA damage repair.

In summary, MCMs can be phosphorylated by multiple kinases, which is critical to maintain the genome integrity from DNA RS and respond DNA damage. However, the mechanistic details for distinct MCM subunits phosphorylation triggering downstream repair components still need to be clarified. Further studies are essential to elucidate undiscovered and putative phosphorylation sites, which is essential for insight into selective approaches to repair DNA damage.

5.2. MCMs Ubiquitination

Protein Ub is a well‐known pathway for target protein degradation. Otherwise, protein Ub also modulates multiple cellular biological processes such as DNA replication, cell cycle checkpoint activation, and DNA repair. Mass spectrometry (MS) results showed that all the MCM proteins in eukaryotes are ubiquitinated in human cells. Of those, MCMs are ubiquitinated by diverse E3 ligases when cells are threatened by DNA damage or RS (Table 1 ). 155

The Kelch‐like ECH‐associated protein 1 (KEAP1) is one crucial candidate of the Cullin3 (CUL3)–RBX1 E3 ligase complex, which ubiquitinates MCM3 in actively proliferating cells. 156 KEAP1‐mediated MCM3 Ub regulates cell cycle progression and genome stability by controlling the MCM2‐7 complex helicase activation. Actually, KEAP1 itself serves as the crucial component in response to oxidative stress, which may be achieved through MCM2‐7 complex chromatin loading.

Recent Ub proteomic analysis revealed that ubiquitin protein ligase E3A (UBE3A) could interact with HERC2 and MCM6 with unknown functions. 157 Apparently, HERC2 is a crucial DNA damage repair factor participating in HR repair at DSB sites. In addition, HERC2, with RNF8, has been shown to promote translesion synthesis (TLS) at stalled replication forks. 158 , 159 Thus, not far to seek, UBE3A‐mediated MCM6 Ub may interact with HERC2 to keep the chromosome stable and further play a role in DNA repair.

During DNA replication termination, CDC49/p97 complex targets polyubiquitinated MCM7 to disengage CMG complex, thus terminating DNA replication. 160 , 161 George et al. 161 supported that polyubiquitylation of MCM7 has a modest effect to interstrand cross‐links (ICLs) repair, which suggests that MCM7 proteasomal degradation may play a more active role in response to DNA damage. Moreover, illustrious HR repair‐associated factor BRCA1 serves as upstream of MCM7 Ub. 162 BRCA1 recruits additional E3 ligases to promote MCM proteins and CMG complex Ub. 163 It is necessary that helicases remove from the damaged DNA after accomplishing recovery. During ICL repair, BRCA1‐mediated CMG Ub assists their disassembly, positioning a distinct regulatory signal to ensure unloading initiation. Thus, Ub‐mediated MCMs unloading provides an appropriate occasion to resolve RS.

The best‐characterized E3 ligase comes from S. cerevisiae , cullin 1 ligase SCF Dia2 , drives CMG ubiquitylation to perform CMG helicase disassembly. However, CMG depolymerizing has various pathways during eukaryotic evolution. 164 Subsequent works indicated that cullin 2 ligase CUL2 LRR1 ‐mediated MCM7 Ub is essential to preserve genome stability during DNA replication termination both in yeast and in human cells. 165 , 166 Further work indicated that two crucial E2 enzymes, UBE2R1/R2 and UBE2G1/G2, connect with CUL2 LRR1 to extend a polyubiquitin chain on MCM7. 165 Ub of MCMs departs from chromatin due to their topological alternation, which may form a functional MCM2‐7 hexamer in their de‐Ub pattern.

Deng et al. 167 suggested that tumor necrosis factor receptor‐associated factor‐interacting protein (TRAIP) acts as ubiquitin ligase associating with the CMG replisome, thus triggering replication fork collapse. Previous work indicated that TRAIP is crucial in regulating normal cell cycle to keep genome stability in eukaryotes. 168 However, Favrizio et al. 166 found that TRAIP‐mediated Ub of MCM7 triggers CMG disassembling in mouse embryonic stem cells. ICL‐induced DNA replication stalling is repaired by endonuclease 8‐like protein 3 (NEIL3) glycosylase and FA pathway. 169 Wu et al. 170 identified stalled replication fork triggers TRAIP‐mediated MCM7 Ub to participate in ICL repair. TRAIP‐mediated MCM7 Ub causes distinct ICL‐repair pathways, NEIL3 recognized short ubiquitin chains to cleave directly, while long ubiquitin chains recognized by p97 complex to trigger FA pathway.

In S. cerevisiae , MCM10 is mono‐ubiquitinated at two distinct lysine sites, subsequently, interacts with PCNA. 171 Expression level of MCM10 is precisely mediated by the CUL4 DDB1 complex. 172 De‐Ub of MCM10 results in hypersensitive to HU owing to dysregulation of the interaction between MCM10 and PCNA.

In summary, K48‐linked MCM7 degradation leads to disassembly of the MCM2‐7 complex, which is critical in DNA replication termination. However, BRCA1‐mediated Ub of MCM7 takes part in HR and ICL repair but not in replication termination. It provides us a novel sight that Ub in different MCM subunits or various sites performs distinct functions. It also possible that specific E3 triggers distinct Ub‐chains to ubiquitinate MCMs for degradation or activation pattern.

5.3. MCMs SUMOylation

SUMO is a protein modifier that plays crucial roles in a wide range of cellular processes, making it essential for the viability of most eukaryotes. SUMOylation is a multistep process modulated by specific E1, E2, and E3 enzymes, like Ub. Compared with Ub, SUMOylation of proteins do not mediate their degradation, modulating their subcellular compartmentalization and reinforcing their stability (Table 1 ). 173 , 174

Previous studies showed that DNA alkylating agents stimulated SUMOylation of MCMs except for MCM3 and MCM7. However, MCM2, 3, 4, and 7 were SUMOylated in response to heat shock in human cell, indicating that MCM SUMOylation may modulate cells against cytotoxic stress. 175

The SUMO‐target ubiquitin ligase Slx5/Slx8 in S. cerevisiae are crucial in modulating DNA repair via SUMOylation repair factors. 176 Coincidentally, Slx5‐based proteomic research revealed that MCM2‐7 complex may be as potential substrates of Slx5/Slx8. These data suggest the Slx5/Slx8‐mediated SUMOylation of MCM2‐7 may take effect during DNA replication and DDR. 177

SUMO modification of MCM3 at K767 and K768 may work together to directly or indirectly promote MCMs loading onto chromatin. Site‐specific mutagenesis of MCM3 K767/768 leads to MCM2‐7 complex disassembly and CMG complex collapse, delaying the chromosomal DNA replication and leading to genome instability. Uniformly, factitious de‐SUMOylation of MCM3 may generate spontaneous DSBs due to incomplete DNA replication, which is quite lethal to cells. 175

S. cerevisiae harbors three SUMO E3 ligases: Siz1, Siz2, and Mms21, which are necessary for controlling intracellular activities. 178 Except for SUMO E3 ligases, SUMOylation is also modulated by SUMO isopeptidase Ulp1 and Ulp2, which performing de‐SUMOylation effect. Ulp2 is inutile for cell viability but necessary for the accumulation of poly‐SUMO chains. 179 de Albuquerque et al. 180 found that loss of Ulp2 aggravates SUMOylation of MCM4 and MCM7, while partially downregulating MCM6 SUMOylation. Mms21, but not Siz1 and Siz2, mediates SUMOylation of MCM3 under HU stimulation, suggesting that Mms21‐dependent SUMOylation of MCM3 might contribute to regulating DNA replication and respond to DNA RS. 180 Siz1/Siz2‐mediated SUMOylation of MCMs has been detected in unperturbed cells, whereas Mms21 preferentially interacting with MCM2 and MCM3. 181 Wei and Zhao found that SUMOylation of MCMs exhibits preference for chromatin‐bound MCM subunits including MCM4, MCM6 and MCM7. SUMOylation of MCM proteins leads to decreased CMG protein levels and inhibits DNA replication initiation. 182

Tian et al. 183 identified a germline variant rs2274110 in MCM10 that confers an inferior survival of esophageal squamous cell carcinoma (ESCC) patients. This functional variant can increase MCM10 SUMOylation resulting in aberrant overexpression, substantially facilitating ESCC progression via fueling DNA over‐replication and genomic instability. These findings underline that PTMs of MCM proteins may serve as potential therapeutic targets in tumor treatment. 183

5.4. MCMs acetylation

Lysine acetylation is a widespread and versatile protein PTM. Indeed, nonhistone protein acetylation is deemed as a key regulatory component in multiple biological processes such as DNA replication, DNA damage repair, autophagy, and metabolism. Several studies revealed that MCM proteins are substrates for acetylation (Table 1 ). 184

MCM3AP acts as an acetyltransferase to acetylate MCM3, which promotes the translocation of MCM3 from the cytoplasm into the nuclei. Moreover, MCM3AP‐mediated acetylation of MCM3 can inhibit DNA replication. 185

HBO1 complexes belong to the MYST family and are major acetyltransferases aiming for histone H4 acetylation in vivo. More recently, HBO1 was deemed to modulate the replication origin of Kaposi's sarcoma‐associated herpes virus. These functional interactions implied a putative function of HBO1 in pre‐RC formation and replication licensing. 186 Lizuka et al. 187 demonstrated that HBO1 significantly acetylates DNA replication‐associated proteins, such as ORC2, MCM2, CDC6, and Geminin. HBO1‐mediated MCM2 might regulate the initiation of DNA replication. 187 During DNA replication initiation step, recruitment of HBO1 to origin by Cdt1 is required for MCM2‐7 complex loading in human cells, which may stabilize the interaction of MCM complex with chromatin. 188 It provides a novel insight that HBO1 may acetylate MCMs to perform conformation alternation, further modulating the DNA replication or DNA damage repair.

SIRT1 is a histone deacetylase that has been implicated in containing chromatin structure and DNA repair, serving as a crucial guard to maintain genomic stability. Samuel et al. 189 demonstrated that deacetylation of MCM10 by SIRT1 is one of the vital regulatory events in preserving genome stability. Moreover, MS and biochemical analysis indicated that twelve lysine residues of MCM10 acetylated by p300 are involved in DNA binding. 189 These results indicated that the dynamic balance of MCM10 acetylation has to be tightly regulated for proper fork initiation and stable genome integrity.

5.5. Other PTMs of the MCM proteins

Protein O‐GlcNacylation is involved in multiple biological processes, especially in stress response. O‐GlcNacylation of proteins is catalyzed by O‐GlcNAc transferase (OGT) to transfer the GlcNAc group onto serine or threonine residues of proteins. Reciprocally, O‐GlcNAcase (OGA) reverses these PTMs by removing the GlcNAc residue. 190 In mammalian cells, O‐GlcNAcylation levels are dynamic alternation during the cell cycle, thus abnormal O‐GlyNAc dynamic cycling disrupts the cell cycle and causes RS. Using a mass‐tagging strategy, Leturcq et al. 191 identified that MCM2‐7 all subunits are O‐GlcNAcylated by OGT in human cells, especially in MCM3, MCM6, and MCM7. Each subunit of MCM2‐7 complex gradually disperses in knockout OGT cells, subsequently departing from chromatin. Thus, it is tempting to speculate that O‐GlcNAcylation of MCMs assists MCM assembly and regulates dynamic balance during DNA damage and DNA replication. 191

Lysine methylation usually occurs in histones, whereas in nonhistones in recent decades. Methylation can alter the conformation of proteins thus changing their function. Xia et al. 192 identified that recombinant Sulfolobus MCM ( sis MCM), an archaeal homolog of MCM2‐7 eukaryotic replicative helicase, is mono‐methylation by aKMT4 in vitro, which is characterized as the first archaeal lysine methyltransferase. Interestingly, MCM methylation (me‐MCM) upregulates MCM complex DNA unwinding ability, modulating their helicase activity. More intriguingly, me‐MCM also enhances heat resistance, which supports that methylation of MCM proteins also impacts protein thermal properties (Table 1 ).

5.6. PCNA Ubiquitination and SUMOylation

During DNA replication, PCNA serves as the pivot to recruit replicative polymerases polε and polδ to perform high‐fidelity DNA synthesis. 193 When replicative DNA polymerase encounters damaged DNA, the progression of the polymerases is blocked, and the replication fork is stalled. If this problem were not be resolved, the replication fork would be collapsed, resulting in cell death. As we summarized above, the blooey replication fork activates a unique DNA repair pathway called postreplication repair (PRR), such as error‐prone translesion DNA synthesis (TLS) and error‐free template switching (TS). 194 Specialized DNA TLS polymerases have been identified in yeast and mammalian, including polymerase η (polη), polymerase ι (polι), and polymerase κ (polκ). For instance, polη mediates efficient and precise TLS past UV‐induced thymine‐thymine CPD (T‐T CPD), whereas resulting in the high frequency of mutations in routine replication progression. 195 Low‐fidelity TLS polymerase causes incorrect nucleotide insertion in normal replication, subsequently stimulating NER, BER, or HR pathways to fix the errors. 196

PCNA is mono‐ubiquitinated at K164 by RAD6–RAD18 E2–E3 complex in response to replication fork stalling. 197 Mono‐ubiquitinated PCNA increases affinity with polη, thus further promoting efficient TLS. 198 Hence, dysregulation of replication and TLS progression cause severe genomic instability and tumorigenesis. Except for the Ub of PCNA, multiple modifications are involved in regulating PCNA functions, thereby impacting DNA replication, DNA repair, and even carcinogenesis.

Ub of PCNA undergoes regulation and control from the chromatin microenvironment by various factors. The chromatin structure could be regulated by PTMs of histone and nucleosome remodeling, which is vital for DNA replication, cell cycle, and DNA damage repair. Mutation of histone H3 and H4 disrupts DNA packaging, resulting in reduced expression of PCNA Ub under methyl methanesulfonate (MMS) or UV irradiation. 199

RAD6–RAD18‐mediated PCNA–K164 mono‐Ub is widely known in a variety of organisms. Meanwhile, polyubiquitination of PCNA has also been observed at K164 residue in yeast and mammal cells, which is essential for inducing error‐free pathways upon damage response. 200 , 201 K63‐linked polyubiquitination of PCNA requires a ternary complex composed of RING‐finger E3 ligase RAD5 and Mms2–Ubc13 complex, protecting cellular DNA against genomic mutations via a TS pathway. 200 , 202 , 203

RNF8 is a crucial E3 ligase in regulating histone Ub during DNA damage repair, while it is also identified as a novel E3 ligase for PCNA. Nevertheless, RNF8 cooperates with distinct E2 such as Ubc15 and Mms2 to activate mono‐Ub and polyubiquitination, respectively. 204 Depletion of RNF8 in medulloblastoma cells significantly suppresses PCNA mono‐Ub under UV damage, which is associated with UV‐induced p53 targeting and checkpoint activation.

In response to stalling DNA replication fork, PCNA could also be modified by SUMOylation at K164 residue, which is commonly observed in yeast and mammals. Furthermore, some reports also supported that PCNA might be SUMOylated at K127 residue and K164 in response to DNA damage. 205 SUMOylation is closely related to Ub that modulates protein coordinated interaction, whereas possibly antagonizes Ub via competing the same residue in substates. 206 In contrast, forceful evidence supported that SUMOylation of PCNA prohibits its Ub and DNA damage repair progression, implying SUMO of PCNA functions on regulating normal DNA replication.

In S. cerevisiae , SUMOylation of PCNA recruit helicase Srs2 via its C‐terminus SUMO‐interaction motif (SIM), which disrupt RAD51 single‐stranded presynaptic filaments, ultimately interrupt HR progression. 207 Furthermore, RFC is required for PCNA SUMOylation, which facilitating PCNA loading onto chromatin. SUMOylation of PCNA also cooperates with PIP, suppressing inappropriate HR. 208 Moreover, Gali et al. 209 revealed that SUMOylation of PCNA could decrease DSBs formation using neutral comets assay, even lower spontaneous recombination frequencies, and enhance damage resistance. Thus, impaired PCNA SUMOylation facilitates DSB formation at stalled replication fork, which highlight its role in preserving genome integrity.

One intriguing perspective supports that SUMOylation could conduct for a signal for Ub. SUMO‐targeting ubiquitin ligases (STUbLs) intervenes protein SUMOylation and Ub of SUMO segments via its SIMs. Mutation of PCNA SUMOylation site K164/K127 and RAD18 SIMs residues directly decrease PCNA Ub level, implying SUMOylation of PCNA facilitates RAD18 to target PCNA. 210 Therefore, it points out that the SUMO and Ub crosstalk may be essential for DNA replication and DNA damage repair (Table 2 ).

Summary of the PCNA modification in response to DNA replication and DNA damage

Modification type	Mediator	Function	Reference
Ubiquitination	RAD18	TLS repair	,
	RAD5	Preserve genome stability and DNA repair	,
	HLTF	TLS repair
	RNF8	Preserve genome stability and DNA repair
SUMOylation		Preserve genome stability and DNA repair	,
		HR repair	, ,
	STUBLs	DNA replication and DNA repair

6. DNA REPLICATION, DISEASE, AND THERAPY

Dysregulation of DNA replication is one remarkable feature of cancer cells associated with tumor progression. 6 Since DNA replication is strictly regulated by CMG complex and multiple polymerases, dysregulation of such replication factors may contribute to abnormal cell cycles causing severe consequences. 211 Accumulating evidence suggests that aberrant expression of CMG serves as reliable diagnostic markers among some cancers. 212 Consistently, MCMs are involved in multiple DDR pathways, such as activation of cell cycle checkpoint, which are also considered as crucial cancer therapeutic strategies.

6.1. MCMs in tumorigenesis and development

Genome instability is a hallmark of cancer. Conventional expression of the MCM2‐7 complex ensures routine DNA replication progression, which is a prerequisite for genome stability. 213 Nevertheless, numerous studies have indicated that both deficiency and overexpression of MCMs are associated with cancer development. Mcm3‐deficient mouse model was used to determine the impact on gene function in hematopoietic stem cells. The results indicated that downregulation of MCM3 results in RS, further leading to fetal anemia during embryonic development. 214 In contrast, MCM6 was overexpressed in clear‐cell renal cell carcinoma. 215 Upregulation of MCM6 also exists in non‐small cell lung cancer and breast cancer with worse survival and higher histological grade. 216 , 217 , 218 The reasons for abnormal MCMs expression remain unclear. There are two possible speculations: (a) CDK‐mediated MCM complex dissociating prevents DNA re‐replication. However, dysregulation of the cell cycle‐dependent kinase CDK permits MCMs constantly binding to each other, resulting in continuous cell division and high expression. 219 , 220 (b) Aberrant DNA replication license system induces abnormal DNA replication, increasing genomic instability, and carcinogenesis. 221 , 222 In addition, spontaneous mutation of MCMs increases chromosome elimination and DNA damage, 223 whereas a more than twofold reduction of MCM protein expression could lead to genomic instability in S. cerevisiae . These findings demonstrated that MCMs are indeed involved in tumorigenesis, but the detailed mechanism is still unclear. 212

In lung cancer, omics data of MCM2 overexpression is analyzed using the Gene Expression Omnibus database, which is associated with large tumor size, different malign degrees, and clinical stages. 224 Using RT‐PCR analysis, except for MCM3 and MCM5, MCMs are upregulated in cervical cancer in vitro and in vivo, which are critical in tumor progression. 225 Spontaneous dominant leukemia ( Sdl ) mice model is a murine model for heritable T cell lymphoblastic leukemia/lymphoma, which harbors a spontaneous mutation in Mcm4 (Mcm4 D573H ). Mcm4 D573H could not alter the total expression of MCM2‐7 complex, whereas it significantly promotes tumor formation. 226 In laryngeal squamous cell carcinoma cells, knockout of MCM4 using siRNA also suppresses cell proliferation and inhibits of tumor progression. 227

MCM7 is regarded as an extensive mark for cancer development. 228 Some studies indicated that MCM7 genome sequence embodies a cluster of miRNAs (miR‐106b, miR‐93, miR‐25), which can downregulate the expression of oncogenes, including p21, E2F1, BIM, and PTEN. 229 PRMT5 acts as one methyltransferase to methylate multiple proteins in histones, 230 which is also deemed a potential target in colorectal cancer development and progression. 231 Recent studies revealed that PRMT5 physically interacts with MCM7 in HCT8 cells, while MCM7 depletion impairs cancer cell migration and invasion. 232 Using TCGA analysis, the expression of MCM7 enhanced by approximately 12% in ESCC. Silencing of MCM7 via siRNA significantly impaired KYSE510 cell proliferation and migration in vitro. 233 Furthermore, miR‐214 targets overexpression of MCM5 and MCM7 in hepatocellular carcinoma (HCC) cells to inhibit cell replication and colony formation. 234

Massive rearrangements are one of the characteristics of aggressive cancer genomes. MCM8, unclassical MCM proteins, is deemed to interrelate with chromosome rearrangement. 235 Knockdown of MCM8 in mice diminished xenografted tumor volume, which implied the critical role of MCM8 in tumor metastasis in vivo. 236

Despite novel and striking findings, additional investigations are still needed to be addressed. Since closely associated with tumorigenesis, MCMs can be used as precise cancer therapy via molecular targets.

6.2. MCMs as diagnostic and prognostic biomarkers

As mentioned above, aberrant expression of MCMs is closely related to tumorigenesis and development. Therefore, MCMs are also used as tumor biomarkers and indicators of prognosis. 237 For example, MCM2 is regarded as a novel proliferation biomarker for oligodendroglioma, 238 ESCC, 239 and breast cancer. 240 , 241 However, the detailed interventional mechanism is different due to MCM2's diversiform role in the distinct process. Knockdown of MCM2 abolishes DNA damage in ESCC cells, interfering with DNA replication in breast cancer cells. Meanwhile, MCM2 is also suggested to be a prognostic marker for some tumors such as renal cell carcinoma, 242 laryngeal carcinoma, 243 , 244 and gastric cancer. 245 , 246 In oral squamous cell carcinoma 247 , 248 and large B‐cell lymphoma, 249 , 250 MCM2 also nominates as a prognostic marker significantly related to malignant progression and the 2‐year survival rate of patients, respectively. In addition, abnormal expression of MCM3 reflects advanced tumor stage and metastatic status in cervical cancer, 251 , 252 breast cancer, 253 , 254 oral squamous cell carcinoma, 255 malignant salivary gland tumors, 256 , 257 and HCC. 258 , 259 Simultaneously, based on the TCGA and GEO analysis, some reports persist that MCM4 mainly serves as a prognostic indicator for HCC, 260 , 261 which is relevant to poor prognosis with MCM4 overexpression pattern. Furthermore, overexpression of MCM4 may also be a diagnostic signal in esophageal cancer, 262 , 263 colorectal cancer, 264 , 265 cervical cancer, 225 , 266 , 267 ovarian cancer, 268 , 269 , 270 , breast cancer, 56 and gastric cancer. 271 , 272 In addition, upregulated expression of MCM5 is mainly related to poor prognosis and malignant status. 115 , 225 , 273 , 274 , 275 Consistent with this notion, overexpression of MCM5 also associates with tumor stages, which appears to be potential diagnostic and prognostic markers in thyroid cancer, 276 , 277 ovarian cancer, 269 , 278 , 279 , 280 bladder cancer, 281 , 282 , 283 and renal cell carcinoma. 284

On the contrary, WGCNA combining with TCGA and GEO analysis identified that exceptional expression of MCM6 reflects pathologic stage of multiple tumors, serving as putative biomarkers for breast cancer, 285 , 286 , 287 gastric cancer, 288 , 289 renal cell carcinoma, 215 , 290 , 291 HCC, 292 , 293 , 294 , 295 and non‐small cell lung carcinoma. 217 , 296 , 297 Meanwhile, MCM7 is widely regarded as an extensive biomarker in multiple tumor types since its overexpression pattern. 233 , 234 , 298 , 299 , 300 , 301 , 302 , 303 , 304 , 305 , 306 , 307 , 308 , 309 , 310 Except for conventional MCM proteins, MCM8, MCM9, and MCM10 are also novel prognostic markers in multiple tumors. In tissues, high expression of MCM8 may act as a valuable prognostic indicator for different cancer therapy, consistent with gastric and cervical cancer. 311 , 312 , 313 MCM9 and MCM10 are associated with additional tumor types such as colorectal cancer, breast cancer, ovarian cancer, and HCC (Table 3 ). 314 , 315 , 316 , 317 , 318 , 319 , 320 , 321 , 322

MCM proteins as diagnostic and prognostic biomarker

Proteins	Cancer type	Reference
MCM2	Oligodendroglioma
	Esophageal squamous cell carcinomas
	Breast cancer	,
	Renal cell carcinoma
	Laryngeal carcinoma	,
	Gastric cancer	,
	Oral cancer	,
	Large B‐cell lymphoma	,
MCM3	Cervical cancer	,
	Breast cancer	,
	Oral squamous cell carcinoma
	Malignant salivary gland tumors	,
	Hepatocellular carcinoma	,
MCM4	Hepatocellular carcinoma	,
	Breast cancer
	Esophageal cancer	,
	Colorectal cancer	,
	Cervical cancer	, ,
	Ovarian cancer	, ,
	Gastric cancer	,
MCM5	Cervical cancer	, , , ,
	Thyroid cancer	,
	Ovarian cancer	, , ,
	Bladder cancer	, ,
	Renal cell carcinoma
MCM6	Breast cancer	, ,
MCM6	Gastric cancer	,
	Renal cell carcinoma	, ,
	Hepatocellular carcinoma	, , ,
	Non‐small cell lung carcinoma	, ,
MCM7	Nonfunctioning pituitary adenomas
	Gastric cancer	, ,
	Hepatocellular carcinoma	, ,
	Meningiomas	, ,
	Prostate cancer
	Oral squamous cell carcinoma	,
MCM8	Gastric cancer
	Cervical cancer
	Bladder cancer
MCM9	Colorectal cancer
MCM10	Ovarian cancer
	Breast cancer	, ,
	Hepatocellular carcinoma	,
	Urothelial carcinoma	,

Compared with the common‐used proliferation biomarker, MCM proteins are more sensitive and particular than PCNA and Ki‐67, 323 accurately reflecting cell proliferation status and predicting prognostic tumor patient's survival rate.

6.3. MCMs as therapeutic target

Since increased DDR preventing cancer cells from effective therapy, MCM2‐7 proteins as intermediates are involved in intricate progression, which may serve as a pivotal role in influencing therapy response.

Conventional chemotherapy is one of the staple cancer treatment strategies, with emerging resistance causing the limited anticancer effects. 324 Previous reviews highlighted that chemotherapy uniting with MCMs knockout approach inhibits tumor cell proliferation. 325 Recently, combination chemotherapies have been widely used in cancer treatment, especially with the knockdown of MCM proteins. Carboplatin‐based chemotherapy is for the initial treatment of ovarian cancer, while carboplatin resistance results in treatment failure. 326 Deng et al. 327 found that knockdown of MCM2 could enhance the carboplatin sensitivity of A2780 cells and increase cells’ UV sensitivity, which may owe to accumulation of damaged DNA and activation of the p53‐dependent apoptotic response. Oxaliplatin or etoposide‐mediated chemotherapy combined with knockdown of MCM7 could reduce the proliferation of colorectal carcinoma cells and induce tumor apoptosis in vitro. 328 In pancreatic ductal adenocarcinoma cells, reduction of MCM4 or MCM7 clearly exhibits more sensitive to gemcitabine and 5‐FU exposure, which may be caused by MCM suppression‐induced RS. 328 Classic hypercholesterolemia curative simvastatin was demonstrated that reduces the expression of MCM7. 329 Thus, it implied that simvastatin combining with chemotherapeutic drugs may be the putative cancer therapy for some chemo‐resistance cancer treatment. Liang et al. 330 demonstrated that simvastatin combines with tamoxifen impaired breast cancer cell proliferation and resulted in apoptosis in vivo and in vitro. Knockout of MCM8 or MCM9 selectively increases cisplatin sensitivity in specific cancer cells such as HCT116 and HeLa cells. 331 , 332 However, siRNA‐mediated MCM8 silencing could not alter the cisplatin sensitivity of normal HFF2/T fibroblasts, indicating MCM8 may act as a molecular target just in cancer cells. Consistent with cisplatin, silencing MCM8 and MCM9 selectively hypersensitizes cancer cells to Olaparib, which may rely on MCM8‐9's role in resolving RS. 333 , 334

Radiotherapy is an alternative cancer therapy known to induce cancer cells’ DNA damage and autophagy to reach the treatment goals, while acquired radio‐resistance disturbs the effectiveness. 335 Next‐generation sequencing of mRNA (RNA‐seq) results revealed that MCM7 is significantly upregulated in radio‐resistant PC‐3 cells after 2Gy IR treatment. Diminishing expression of MCM7 might increase radiotherapy response in prostate cancer. 336 Consistent with this notion that upregulated expression of MCM2 is involved in the radio‐resistant cervical cancer cell, indicating that MCM2 is one potential regulatory factor in increasing radio‐sensitivity in cancer treatment. 337 As mentioned above, MCM3 is one prognosis marker for HCC with high expression in vitro and in vivo. Using MTT and TUNEL methods, low MCM3 expression HCC cell line performs low growth, whereas high MCM3 expression induces lower apoptosis under radiotherapy. In addition, overexpression of MCM3 further promotes HCC cell radio‐resistance, revealing MCM3 prevents HCC radiotherapy efficiency via activating NF‐κB pathway. 259

Specific small molecule inhibition for MCM proteins is an additional invaluable approach for various cancer treatments. 338 There are three typical MCMs‐based small molecule inhibitors with potential chemotherapeutic effects: (a) Enzyme inhibitors such as DNA helicase‐targeting small molecule inhibitors. 339 Ciprofloxacin targets MCM2‐7 complex to block the helicase activity, further inhibiting cell proliferation. 340 , 341 (b) The inhibitors prevent interaction among MCM subunits. 112 , 342 , 343 (c) The inhibitors regulate the expression of MCM proteins. 344 Widdrol could downregulate the expression of MCM proteins to inhibit cancer cell proliferation in G1 phase. 345 , 346 In addition, trichostatin A targets MCM2 to inhibit its expression. 347 , 348 Recent studies revealed that Breviscapine downregulates the expression of MCM7 and impairs tumor progression in prostate cancer via activating DNA damage‐induced apoptosis (Table 4 ). 349

MCM proteins as therapeutic target in different therapeutic scheme

Therapy	Cancer type	Target protein	Therapeutic scheme	Reference
Chemotherapy	Ovarian cancer	MCM2	Carboplatin	,
	Colorectal carcinoma	MCM7	Oxaliplatin
	Pancreatic ductal adenocarcinoma	MCM4, MCM7	Gemcitabine/5‐FU
	Breast cancer	MCM7	Tamoxifen/Simvastatin	, ,
	Ovarian cancer	MCM8, MCM9	Olaparib	,
Radiotherapy	Prostate cancer	MCM7	IR
	Cervical cancer	MCM2	IR
	Hepatocellular carcinoma	MCM3	IR
Small molecule inhibitor		MCM2‐7	Ciprofloxacin
		MCM2‐7	Widdrol	,
		MCM2	trichostatin A	,
		MCM7	Breviscapine

6.4. GINS and Cdc45 as prognostic markers and the target for therapy

Except for MCMs, concurrent overexpression of GINS and Cdc45 are also observed in various cancers. Since Psf1 promoter activity is related with 17β‐estradiol (E2)‐based estrogen receptor pathway, aberrant expression of Psf1 might be a signal in breast cancer. Nakahara et al. 350 revealed that expression of Psf1 is remarkably increased in breast cancer both in vivo and in vitro, whereas siRNA‐mediated depletion of Psf1 inhibits DNA replication and cell growth. This evidence provides that Psf1 could be deemed as a novel breast cancer biomarker and therapeutic benefit for breast cancers with overexpression of Psf1. 350 Moreover, uprelated expression of Psf1 is also detected in non‐small cell lung cancer, which implies Psf1 as a prognostic biomarker and potential target for lung cancer therapy. 351 , 352 Using the tumor cell xenograft model, Nakahama et al. found higher expression of Psf1 is correlative with higher proliferative ability and metastatic capability, implicating Psf1 in tumorigenesis and its conceivable role as a therapeutic target. Furthermore, anomalous expression of Psf1 also exists in prostate cancer and HCC, 353 , 354 significantly correlated with tumor grade and clinical stage.

Using cDNA microarray analysis, Psf2 is frequently upregulated in cholangiocarcinoma, while knockdown of Psf2 drastically reduces cell proliferation and inhibits cell growth. 355 Furthermore, obvious upregulation of Psf2 is detectible in cervical cancer cell, whereas downregulation of Psf2 inhibits cell multiplication and tumorigenic ability. 356 Combined with the observations, Psf2 might be a novel and valuable prognostic biomarker for ovarian cancer and leukemia, 357 , 358 subsequently an underlying molecular target for cancer diagnosis and treatment. In triple negative breast cancer (TNBC) cell lines, similar with Psf1, the expression of Psf2 is also enriched correlated with the advanced stages of tumor. Intriguingly, silencing of Psf2 decreases the expression of matrix metallopeptidase 9, which is necessary for tumor invasion, hence suppress tumor cell migration and invasion. 359

Aberrant expression of Psf3 in colon carcinoma cell line associates with tumor cell proliferation and tumor progression, simultaneously, the similar results have also been found in non‐small cell lung cancer, lung adenocarcinoma, and colorectal cancer patients. 360 , 361 , 362 , 363 Finally, abundant Sld5 expression is closely related to bladder cancer and gastric cancer, since defect of Sld5 causes severe tumor cell proliferation disorder and inhibits cell growth. 364 , 365

Upregulated expression of Cdc45 is also a predictor for multiple cancers, including breast cancer, leukemia, lung cancer and osteosarcoma. 366 , 367 Using the Cancer Genome Atlas, prominent overexpression of Cdc45 is discovered in papillary thyroid cancer tissue, which impacts tumor sizes and cancer stages. 368 In tongue squamous cell carcinomas (SCC), higher expression of Cdc45 with severe lymph node status is observed in malignant tumor than mild precancerous epithelial dysplasia, which implies its role in distinguish precancerous dysplasia from SCC. 369 Myc is one crucial factor in regulating cell growth and tumorigenesis, overexpression of Myc enhances the Cdc45 DNA binding ability and replication fork stalling, which indicates Myc‐induced RS collaborates with Cdc45 in tumor development. 370 , 371

In summary, excessive expression of CMG is generally related with tumor size and malignancy but to varying extents, depending on the type of tumor. Thus, except for MCMs, GINS and Cdc45 could also serve as diagnostic and prognostic biomarkers for multiple tumors, which also as a druggable target to treat cancers (Table 5 ).

GINS, Cdc45, and PCNA as prognostic biomarker

Proteins	Cancer type	Reference
Psf1	Breast cancer
	Non‐small cell lung cancer	,
	Prostate cancer
	Hepatocellular carcinoma
Psf2	Cholangiocarcinoma
	Cervical cancer
	Ovarian cancer
	Leukemia
	Breast cancer
Psf3	Non‐small cell lung cancer	,
	Lung adenocarcinoma
	Colorectal cancer
Sld5	Bladder cancer
Sld5	Gastric cancer
Cdc45	Breast cancer
	Leukemia
	Lung cancer	,
	Osteosarcoma
	Thyroid cancer
	Tongue squamous carcinoma
PCNA	Lung cancer
	Prostate cancer
	Breast cancer
	Colorectal cancer
	Cervical cancer	,
	Esophageal squamous carcinoma
	Hepatocellular carcinoma

Cancer immunotherapy is a novel biological treatment for malignant tumors, which could be collaborated with traditional radiotherapy and chemotherapy to improve the curative rate. Cancer immunotherapy elicits the immune system by identifying specifically expressed molecules on the tumor surface, thereby eliminating the cancer cells. 372 Cytotoxic T lymphocytes (CTLs) are specific T cells with potent nocuity to cancer cells, which recognizing cancer‐specific antigenic peptides human leukocyte antigen (HLA). Though mass spectrometric analyses and bioinformatic analysis, Yoshida et al. 373 isolates a Psf1‐derived peptide presented by HLA. Moreover, they found no other cancer vaccine target proteins except for Psf1. Detailed peptide is verified using the mouse model, suggesting Psf1 79–87 peptide induces CTL response in vitro and in vivo, which provides a novel cancer immunotherapy for targeting cancer stem cells. Previously, the similar approach was utilized in Cdc45, demonstrating that strongly immunogenic Cdc45‐derived peptides stimulated CTLs to be reactive to lung cancer cells. 373

6.5. PCNA as prognostic markers and the target for therapy

Due to its role in cell proliferation, PCNA is deemed as the tumor marker for diagnosis and patient prognosis. Overexpression of PCNA is observed in lung cancer in vitro and in vivo, while silencing of PCNA reduces cell invasion ability and 95D cells proliferations. 374 Identical results also occur in prostate carcinoma and breast cancer that PCNA connects with pathological stage and cellular grade, suggesting PCNA might be a crucial prognostic indicator of malignant tumors. 375 , 376 Furthermore, upregulated PCNA is associated with various digestive system tumors including colorectal carcinoma, ESCC, and HCC (Table 5 ). 377 , 378 , 379

PCNA is a critical factor in DNA replication and DNA damage repair. Hence, PCNA is a target for designing antiproliferation and anticancer drugs. Since multiple PTMs of PCNA interrupt its chromatin binding ability, developing therapeutics of modified PCNA will be conducive to target cancer cells. Previous research found that Y211 phosphorylation of PCNA is a crucial event to preserve the PCNA stability, promoting DNA damage repair and DNA synthesis. Therefore, mutant of Y211 phosphorylation inhibits tumor cells proliferation including prostate cancer and breast cancer. 380 , 381 In addition, specific small molecular inhibitors targeting PTMs of PCNA are also identified to disrupt cell proliferation and enhance chemosensitivity or radiosensitivity. RAD6 selective small molecular inhibitor SMI#9 leads to PCNA mono‐Ub defect and mitochondrial function reduction, suggesting RAD6 inhibitor serves as a promising strategy for TNBC treatment. 382

Although PTMs of PCNA have several implications in carcinogenesis; however, the detailed antitumor mechanism needs further investigation. Moreover, novel inhibitors or strategies inhibiting tumor development or proliferation via targeting PTMs of PCNA require identification.

7. CONCLUSION AND PERSPECTIVES

Numerous proteins perform DNA replication to maintain genetic information transmission from the parental generation to the next generation. In line with this, DNA replication factors are strongly associated with DDR to ensure genome integrity. Abnormal expression of DNA replication proteins results in genomic instability and performs a surprising diversity of symptoms. In this review, we outline the DNA replication mechanism and review the distinct roles of replication factors in DNA replication, DDR, and tumorigenesis.

Increasing publications revealed abundant replication factors are involved in DDR upon RS. Dynamic status of these proteins is regulated by some kinases, such as ATR and ATM, while multiple PTMs also modulate their functions and architecture. However, PTM‐mediated structure alternation may also contribute to DNA topological change to assist chromosomal rearrangement. 383 As we summarized, PTMs of MCMs and PCNA impact critical integration of DNA replication, DDR, and even cancer therapy. Nevertheless, the temporospatial modification crosstalk and definite modification sites still need deep investigation. It will be fascinating to clarify the crosstalk among distinct PTMs and map a trenchant signal network of DNA replication licensing and DNA damage repair.

Nevertheless, loss and mutation of the DNA replication factors is also the source of various disorders. In mammals, loss of function of polη leads to a hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by a high frequency of skin cancer. 384 In addition, mutations in ORC complex cause the developmental disorder Meier‐Gorlin syndrome and Wolf‐Hirschhorn syndrome. 385 These atypical diseases with defects in DNA replication are still a tough job to explore in the future.

Dysregulation of DNA replication in cancer cells causes carcinogenesis with aberrant expression of replication proteins. Thus, such replication factors are closely related to tumorigenesis and development, acting as diagnostic and prognostic biomarkers among multiple tumors. Based on numerous biological research, overexpression of CMG and PCNA disturbs routine cell proliferation and regular DNA damage repair, promoting tumor development. Therefore, target therapy may be a potential approach to cancer treatment. In this review, we also arranged the current understanding of combination anticancer strategies with knockdown of replication factors. With the rapid evolution of pharmaceutics, small molecule inhibitions targeting these proteins are also designed for clinical application. Considering the PTMs of these proteins are also involved in DNA replication and their activity, PTMs of replication proteins may be a putative field to design associate small molecule inhibitors.

AUTHOR CONTRIBUTIONS

Hao‐yun Song was responsible for the manuscript. Hao‐yun Song designed the project in collaboration with De‐gui Wang and Rong Shen. Hao‐yun Song wrote the manuscript. Ya‐nan Guo, Hamid Mahasin, Rong Shen, and De‐gui Wang revised the manuscript. All authors have read and approved the final manuscript.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ETHICS STATEMENT

Acknowledgments.

This work was financially supported by National Natural Science Foundation of China (No. 82071695 and 82060535), Natural Science Foundation of Gansu Province (No. 21JR7RA450), Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT320005), and Fundamental Research Funds for the Central Universities (lzujbky‐2022‐it14).

Song H‐Y, Shen R, Mahasin H, Guo Y‐N, Wang D‐G. DNA replication: Mechanisms and therapeutic interventions for diseases . MedComm . 2023; 4 :e210. 10.1002/mco2.210 [ CrossRef ] [ Google Scholar ]

Contributor Information

Hao‐Yun Song, Email: nc.ude.uzl@02yhgnos .

Rong Shen, Email: nc.ude.uzl@rnehs .

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9.2: DNA Replication
This is accomplished by the process of DNA replication. The replication of DNA occurs during the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis. The elucidation of the structure of the double helix provided a hint as to how DNA is copied. Recall that adenine nucleotides pair with thymine nucleotides ...
DNA Replication Steps and Process
The replication process follows several steps involving multiple proteins called replication enzymes and RNA, or ribonucleic acid. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of the cell cycle. Before this phase, also known as the synthesis stage, the cell passes through a preparation phase ...
Essay on DNA Replication
The process of DNA replication takes place under the control of DNA polymerase. In other words, the process is catalized by the polymerase enzyme. In eukaryotes, four types of polymerase enzymes, viz. alpha, delta, gamma and epsilon are used. ... Essay # Mechanism of DNA Replication: The semi-conservative model (mechanism) of DNA replication ...
DNA Replication: Enzymes, Mechanism, Steps, Applications
DNA replication is the process of producing two identical copies of DNA from one original DNA molecule. DNA is made up of millions of nucleotides, which are composed of deoxyribose sugar, with phosphate and a base. The complementary pairing of these bases keeps the double strands intact. So, to make two copies of one DNA, these hydrogen bonds ...
9.2 DNA Replication
This is accomplished by the process of DNA replication. The replication of DNA occurs during the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis. The elucidation of the structure of the double helix provided a hint as to how DNA is copied. Recall that adenine nucleotides pair with thymine nucleotides ...
DNA replication
The replication fork is a structure that forms within the long helical DNA during DNA replication. It is produced by enzymes called helicases that break the hydrogen bonds that hold the DNA strands together in a helix. The resulting structure has two branching "prongs", each one made up of a single strand of DNA.
14.3C: DNA Replication in Eukaryotes
Figure 14.3C. 1 14.3 C. 1: Replication Fork Formation: A replication fork is formed by the opening of the origin of replication; helicase separates the DNA strands. An RNA primer is synthesized by primase and is elongated by the DNA polymerase. On the leading strand, only a single RNA primer is needed, and DNA is synthesized continuously ...
DNA Replication: Steps, Process, Diagram And Simple Explanation
Initiation, elongation and termination are three main steps in DNA replication. Let us now look into more detail of each of them: Step 1: Initiation. The point at which the replication begins is known as the Origin of Replication (oriC). Helicase brings about the procedure of strand separation, which leads to the formation of the replication fork.
Cells Can Replicate Their DNA Precisely
Replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. DNA replication is one of the most basic processes that occurs within a cell.
2.5: DNA Replication
DNA Replication. Having established some basic structural features and the need for a semi-conservative mechanism it is important to understand what is known about the process and to think about what questions one might want to answer. Since DNA replication is a process we can invoke the "energy story" to think about it.
DNA Replication
DNA has directionality that can run either 3′-5′ or 5′-3′ based off of the carbons in the sugar group. The two strands of DNA in the double helix must run opposite to each other in an anti-parallel fashion. Therefore, if the first strand starts at the 3′ end and finishes at the 5′ end, then the second strand must run opposite, starting at the 5′ end and finishing at the 3′ end.
PDF DNA Replication
DNA Replication 9 Living cells must be able to duplicate their entire set of genetic instructions every time they divide. Likewise, multicellular organisms must be able to ... how this replication process is carried out accurately and rapidly. DNA replication is semi-conservative As we saw in Chapter 8, the self-complementarity of DNA suggested ...
16.4
DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand.
DNA Replication (A-level Biology)
DNA replication occurs before the cell divides. DNA replicates itself during the S phase of the cell cycle so that each daughter cells has a copy of the DNA after cell division. DNA replication mean that parents can pass their DNA to their offspring. This passing of DNA and the genetic information stored in DNA is known as " Genetic ...
replication
replication. DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. Replication is an essential process because, whenever a cell ...
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Semi-Conservative DNA Replication
Replication is the process by which a cell copies its DNA prior to division. In humans, for example, each parent cell must copy its entire six billion base pairs of DNA before undergoing mitosis ...
Recent advances in understanding DNA replication: cell type-specific
Introduction. DNA synthesis occurs during the S phase of the cell cycle and is ensured by the replisome, a molecular machine made of a large number of proteins acting in a coordinated manner to synthesize DNA at many genomic locations, the replication origins 1.Replication origin activation in space and time (or replication program) is set by a sequence of events, starting already at the end ...
Biochemistry, DNA Replication
The existence of cell division implies that there is a mechanism that replicates DNA and supplies identical copies for the daughter cells while still maintaining an accurate representation of the genome. This mechanism, known as DNA replication, occurs in all organisms and allows for genetic inheritance. It can occur in a short period, copying up to approximately ten to the 11th power (10^11 ...
14.4: DNA Replication in Prokaryotes
RNA primers are removed by exonuclease activity. Gaps are filled by DNA pol by adding dNTPs. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds. Table 14.4.1 14.4. 1 summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.
DNA Replication Process with Diagrams Class 12
The DNA replication in eukaryotes is similar to the DNA replication in prokaryotes. However, the initiation process is more complex in eukaryotes than prokaryotes. In eukaryotes, there are multiple origins of replication present. A pre-replication complex is made with other initiator proteins. The process is entirely the same but the enzymes ...
DNA Replication as a Semiconservative Process Essay
DNA Replication as a Semiconservative Process Essay. The process of DNA replication has been studied extensively as the pathway to understanding the processes of inheritance and the possible platform for addressing a range of health issues occurring as a result of DNA mutations. Get a custom essay on DNA Replication as a Semiconservative Process.
DNA replication: Mechanisms and therapeutic interventions for diseases
DNA replication is an intricate process with a coordinated interplay of multiple proteins. As we summarized, each step of DNA replication must be strictly regulated to preserve genome integrity, while internal or external DNA‐damage agent always threatens DNA replication to activate DDR system. Meanwhile, dysfunction of DNA replication and ...
7.2: Semi-Conservative DNA Replication
One of the most important concepts of DNA replication is that it is a semi-conservative process (Figure 7.2.7 7.2. 7 ). This means that every double helix in the new generation of an organism consists of one complete "old" strand and one complete "new" strand wrapped around each other. This is in contrast to the two other possible ...

DNA Replication Steps and Process

Key Takeaways

What Is DNA and Why Does It Replicate?

DNA Structure

Replication Preparation and Beginning

Step 1: Replication Fork Formation

Replication Begins

DNA Replication: Elongation

Step 3: Elongation

Step 4: Termination

Replication Enzymes

DNA Replication Summary

Essay on DNA Replication | Genetics

Essay # Definition of DNA Replication :

Essay # Mechanism of DNA Replication :

Essay # Evidences for Semi-Conservative DNA Replication :

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DNA Replication: Enzymes, Mechanism, Steps, Applications

Mechanism of DNA Replication

Enzymes and Proteins Used in DNA Replication

Steps in DNA Replication

Step 2: Initiation

Step 3: Elongation

Step 4: Termination

Okazaki Fragments

Replication Fork Formation and its function

Leading Strand

The Lagging Strand

Applications of DNA Replication

DNA Replication Stress

Similarities between Prokaryotic and Eukaryotic DNA Replication

Eukaryotic vs. Prokaryotic DNA Replication (11 Major Differences)

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9.2 DNA Replication

DNA Replication in Eukaryotes

Visual Connection

Telomere Replication

DNA Replication in Prokaryotes

Link to Learning

How Does DNA Replication Occur? What Are The Enzymes Involved?

Mitosis Vs Meiosis – How Does Cell Division Work?

What Is A Deletion Mutation?

Does Human DNA Change With Time?

What Is Cytokinesis?

What Is DNA And How Does It Work?

Chromatin: Structure And Function Within A Cell

Nuclear Fission v Nuclear Fusion: Differences and Similarities Explained

Germination: How Does A Seed Become A Plant?

Who's J. Robert Oppenheimer And Why Is He A Big Deal?

What are Mutations and what are the different types of Mutations?

Cellular Respiration: How Do Cell Get Energy?

What is Evolution: A REALLY SIMPLE and Brief Explanation

DNA Replication

DNA Nucleotides

DNA Structure

DNA Replication Is Semiconservative

DNA Replication Process

Proteins in DNA Replication

The Replication Bubble

Replicating the Leading Strand

Replicating the Lagging Strand

Prokaryotes vs. Eukaryotes

Telomeres Affect Cell Age

DNA Repair and Damage

Environmental Damage

Bibliography

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DNA Replication (A-level Biology)

The Process of DNA Replication

2) Semi-Conservative Replication

3) DNA Polymerase (Condensation Reactions)

Mechanism of DNA Polymerase

DNA polymerase Works in the 5′ to 3′ direction

DNA polymerase reads and DNA ligase catalyses

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