DNA Replication: Definition, Enzymes, Steps, Applications

DNAreplication 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

Enzymes and Proteins used in DNA Replication

Nucleases

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聚合酶的酶,这种酶是负责任的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 ofE.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

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 ofHydrogen bondsbetween annealed nucleotide bases.

DNA primase

Primase is an enzyme that is capable to synthesize short stretches ofRNAsequences 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:

Nucleases
DNA Polymerase
DNA ligase
DNA helicase
DNA primase
DNA topoisomerase
Single strand binding proteins

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 3′ to 5′ direction (leading strand) and the other strand is oriented to 5′ to 3′ 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 3′ to 5′ direction towards the replication fork and is referred to as leading strand and the other strand runs from 5′ to 3′ away from the replication fork and is referred to as lagging strands.
对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.

DNA Replication. Created with BioRender.com

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 3′ to 5′ 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.
最终,复制叉后终止minating 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
Initiation
Elongation
Termination

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.

Eukaryotic vs Prokaryotic DNA Replication (11 Major Differences)

S.N.	Eukaryotic DNA Replication	Prokaryotic DNA replication
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.	DNA gyrase(Telomerase) is needed.	不需要DNA促旋酶(端粒酶)。

References

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