Enzymes used in DNA replication

DNA replication is a many-step biological process that produces two identical copies from a single parent DNA molecule. It is an important aspect of biological heredity and is found in all living species, both prokaryotes and eukaryotes. The process requires the involvement of enzymes, protein components, and metal ions.

This Story also Contains

1. What is DNA Replication?
2. Structure of DNA
3. Role on Replication
4. Stages of DNA Replication
5. Key Enzymes Involved in DNA Replication
6. Recommended video for Enzymes in DNA replication
7. MCQs on Enzymes in DNA Replication

Watson and Crick proposed the double helical structure of DNA in 1953. They also proposed that the two strands of DNA separate and serve as templates for the formation of daughter strands during replication. Each DNA molecule would have 1 parental DNA and 1 newly synthesised daughter strand. This demonstrates that DNA replication is a semi-conservative process. Enzymes used in DNA replication are a topic of the chapter Molecular Basis of Inheritance. It is an important chapter in the Biology subject.

What is DNA Replication?

Replication of DNA 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 reproduction of living organisms. Several key enzymes play their parts during replication to ensure the DNA is accurately and efficiently duplicated.

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 on 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

The process of replication of DNA includes 3 steps: initiation, elongation and termination. The semi-conservative nature was established by the Meselson and Stahl experiment. Various enzymes are also involved in the process. The process is described in detail below-

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

Various enzymes are utilised during the process of replication of DNA. Enzymes such as helicase, primase, polymerase, ligase, etc. are used during the process of 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 (SSBPs)

• 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.

• SSBPs 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.

RNase H

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

Telomerase

• Telomerase is an enzyme that lengthens 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.

• They also fight against DNA damage.

Recommended video for Enzymes in DNA replication

MCQs on Enzymes in DNA Replication

Q1. Assertion: DNA helicases are enzymes which separate the double stranded DNA strands for replication.

Reason: Topoisomerase I and II creates cut and pass through the double- stranded DNA.

Option 1: Both Reason and Assertion are true.

Option 2: Reason is true, but the assertion is false.

Option 3: The assertion is true, but the reason is false.

Option 4: Both Reason and Assertion are false.

Correct answer: 3) The assertion is true, but the reason is false.

Explanation:

Nuclear enzymes known as topoisomerases are crucial for DNA replication, chromosome segregation, transcription and recombination. Topoisomerases come in two main varieties; type I enzymes cut single-stranded DNA, while type II enzymes pass double-stranded DNA. To release the topological tensions created during transcription, topoisomerases are required.

Hence, the correct option is 3) The assertion is true, but the reason is false.

Q2. Choose the suitable answer

The role of DNA helicase enzyme is to_______

Option 1: Ligate the phosphodiester bonds.

Option 2: Unwind the DNA strands

Option 3: Catalyze degradation of RNA

Option 4: Synthesizing polymers of nucleic acids

Correct answer: 2) Unwind the DNA strands

Explanation:

It is believed that all organisms require the class of enzymes known as helicases. Their primary job is to unravel an organism's genetic code. Using the energy from ATP hydrolysis, helicases are motor proteins that travel in one direction along a nucleic acid phosphodiester backbone to separate two hybridized nucleic acid strands. An extremely conserved class of enzymes known as DNA helicases unwinds DNA. All procedures involving access to single-stranded DNA, including RNA transcription, DNA replication, and DNA repair and recombination, are carried out by them.

Hence, the correct answer is Option 2) Unwind the DNA strands

Q3. Which of the following covalently connects segments of DNA?

Option 1: Ligase

Option 2: Polymerase

Option 3: Helicase

Option 4: Gyrase

Correct answer: 1) Ligase

Explanation:

Ligase: This enzyme facilitates the creation of covalent connections between neighboring DNA segments' sugar-phosphate backbones. Because it seals the nicks between the freshly formed strands, it is essential for processes like DNA replication and repair.

DNA polymerase: By appending nucleotides to an expanding chain, this enzyme creates new DNA strands. Although it is essential for DNA replication, it does not directly covalently join DNA segments; instead, it adds nucleotides to the chain, whereas ligase does so.

Helicase: During replication and other procedures, this enzyme unwinds double-stranded DNA, separating the two strands. DNA segments are not covalently bonded by it.

DNA Gyrase: This enzyme, a kind of topoisomerase, helps to release the tension that unwinding causes in the DNA helix.

Hence, the correct answer is option 1) Ligase

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