Enzymes used in DNA replication

What Is DNA Replication?

DNA replication is a process by which the cell makes an exact copy of its DNA. In this way, genetic material of exactly the same nature is passed on to each new cell. It is pivotal for the growth, development, and maintenance of living organisms. Several key enzymes play their parts during replication to ensure the DNA is accurately and efficiently duplicated.

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Structure Of DNA

DNA, or deoxyribonucleic acid, is structured as a double helix. It was a model by James Watson and Francis Crick that had two very long strands of nucleotides twisted against each other. Each nucleotide is usually composed of one sugar molecule, a deoxyribose sugar, one phosphate group, and one nitrogenous base—a purine or a pyrimidine: adenine A; thymine T; cytosine C; and guanine G. The sugar-phosphate backbone is the outer structure of the helix, and the nitrogenous bases pair up—A with T, C with G—via hydrogen bonds.

Role In Replication

The double helix unwinds and each strand serves as a template for a new complementary strand. The process depends on base-pairing principles: A with T, and C with G. The DNA strands have directionality. One end of the strand is called the 5' (five prime), and the other is called the 3' (three prime). Adjacent nucleotides are respectively linked by 5'-3' phosphodiester bonds. DNA replication occurs in a 5' to 3' direction.

Stages Of DNA Replication

1. Initiation:

• Replication begins at specific locations that are found in DNA within the origin of replication.

• The DNA double helix is unwound by an enzyme called helicase, which provides the replisome with a region to act on and creates a replication fork.

• The initiator proteins identify and bind to the origin of replication; this serves as a marker of where replication actually starts.

2. Elongation:

• DNA polymerase synthesises new strands by adding nucleotides that are complementary to template strands.

• The leading strand is continuously synthesised in a 5′ to 3′ direction.

• The lagging strand is synthesised in a discontinuous fashion, resulting in short fragments called Okazaki fragments.

• Periodically, primase lays down RNA primers on the lagging strand and initiates the synthesis of each Okazaki fragment.

3. Termination:

• The process of replication is terminated when the replication forks finally meet and the synthesis of the new strands of DNA is complete.

• DNA ligase joins the Okazaki fragments on the lagging strand to form one continuous strand.

• Termination sequences or proteins help to disintegrate the replication apparatus once the replication is complete.

Replication Fork And Bubble

The replication fork refers to the Y-shaped region of the DNA in which there is a division into two different strands and where there is replication. A replication bubble will form as the DNA helix opens at several origins of replication, continuing along other areas of the DNA at the same time.

Key Enzymes Involved In Dna Replication

The key enzymes in DNA replication are:

Helicase

• DNA replication needs to occur in the nucleus, but DNA is very long. Helicase unwinds the double helix of DNA, breaking the hydrogen bonds that hold the nitrogenous base pairs together.

• It binds to the origin of replication and moves along the DNA, separating the two strands to expose the replication fork.

Single-Strand Binding Proteins (Ssbs)

• These bind to the single-stranded DNA exposed by the action of helicase and prevent the strands from re-annealing.

• They stabilise the single-stranded DNA and protect it from nucleases.

• SSBs prevent the formation of secondary structures that can impede the replication process.

Primase

• Primase synthesises short RNA primers on DNA template strands.

• These RNA primers provide a starting point for DNA polymerase to begin DNA synthesis.

• It is the primase activity which is responsible for initiating the synthesis of both the leading strand and lagging strand.

Dna Polymerase

• DNA polymerase adds nucleotides to the growing DNA strand complementary to the template strand.

• DNA Polymerase III in prokaryotes and DNA Polymerase δ and ε in eukaryotes are primarily responsible for DNA synthesis.

• This DNA polymerase has an exonuclease activity, which serves to remove wrongly paired nucleotides and replace them with correctly paired ones.

• This proofreading function allows the replication of DNA with a high degree of fidelity.

Sliding Clamp

The sliding clamp is a ring-shaped protein that encircles DNA, binding to DNA polymerase and increasing its processivity—the ability to synthesise long stretches of DNA without dissociating.

DNA Ligase

• DNA ligase seals Okazaki fragments on the lagging strand into continuous lengths by forming phosphodiester linkages between the 3'-OH end of one fragment and the 5'-P end of the next one.

• It seals nicks in the sugar-phosphate backbone, thereby generating a continuous strand.

• The covalent bonds forming the continuous strands of DNA between adjacent nucleotides are fashioned by DNA ligase using ATP.

Topoisomerase

• Topoisomerase prevents supercoiling and tangling of DNA ahead of the replication fork.

• Topoisomerase I: Relieves torsional strain by transiently cutting one DNA strand.

• Topoisomerase II: Relieves supercoiling by transiently cutting both strands.

Other Enzymes And Factors

RNase H

• RNase H removes RNA primers from the newly synthesized DNA.

• It degrades the RNA strand of RNA-DNA hybrids, providing a template for DNA polymerase to fill in with DNA.

• RNase H ensures that there are no RNA sequences left in the newly synthesized DNA.

Telomerase

• Telomerase is an enzyme that lengths telomeres, those repetitive nucleotide sequences that cap the ends of the chromosomes.

• In holding up chromosome length and stability, the activity of telomerase is important.

• It is particularly active in stem cells and germ cells.

• Telomerase has striking implications for ageing and cancer. If its action counteracts telomere shortening, this will be a hallmark of cellular ageing.

DNA Repair Enzymes

• DNA repair enzymes correct the errors that occur during replication and thus also fight against DNA damage.

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Examples And Mechanisms

• Exonucleases: The enzymes remove mismatched nucleotides.

• DNA Glycosylases: They remove the damaged bases and consequently initiate base excision repair.

• Nucleotide Excision Repair: It is a process that removes bulky DNA lesions. For example, thymine dimers occur because of the action of UV radiation.

• Mismatch Repair: The system corrects errors that may have bypassed the proofreading activity of the DNA polymerases during replication.

Conclusion

The process of DNA replication is central to life. It assures an identical duplication of genetic material. In its process, it involves the interplay of different enzymes, which perform specific functions. Helicase unwinds the DNA, SSBs stabilise the single strands, primase synthesises RNA primers, DNA polymerase adds nucleotides, the sliding clamp enhances polymerase activity, DNA ligase joins the Okazaki fragments, and topoisomerase prevents supercoiling. Other factors, for instance, RNase H and telomerase play a vital role in primer removal and telomere extension respectively. Knowing and understanding these enzymes is of importance primarily to students and, more importantly, to anyone interested in the molecular mechanism of life.