Replication of DNA

Replication of D.N.A:

The formation of carbon copy or replica of D.N.A from parental D.N.A is called replication of D.N.A or duplication of D.N.A.The replication takes place inside the nucleus of eukaryotes in the S-phage of the cell cycle and in the cytoplasm of the prokaryotic cell. Watson and Crick not only proposed the three-dimensional model of D.N.A but also suggested the mechanism of D.N.A replication. In replication, both D.N.A strands uncoil and separate. Each strand acts as a template strand for the formation of a new and complementary D.N.A strand. The template and newly synthesized strand form a new double strand which is identical to the parent D.N.A molecule.

Methods of Replication:

Del Bruck suggested three theoretical methods for the replication of Watson and Crick model of D.N.A. They are as follows :

1)Conservative mode of replication:-

In this type of replication, double-stranded parent D.N.A synthesizes replica or new D.N.A and then it remains conserved as it is.

2) Dispersive mode of replication:-

In this type of replication, the parental D.N.A synthesizes two daughters D.N.A after which it(parental D.N.A) gets disintegrated.

3)Semi-conservative mode of replication:-

Meselson and Stahl by using N¹⁴and N¹⁵ isotopes of Nitrogen confirmed that the replication of D.N.A inE.coli bacterium is semi-conservative.

In this method, two strands are separated by breaking hydrogen bonds of their nitrogen bases. Both the strands of DNA function as master or template strand which synthesize new or complementary strands from the medium. As a result, two new DNA molecules are synthesized in which one strand is original and another strand and the other strand is newly assembled. Thus, a newly synthesized DNA is also called hybrid DNA.

Basic requirements for DNA replication:

The mechanism of DNA replication is a complex and involves many steps. Several enzymes, protein factors and metal ions are required for this process.

A) DNA helicase: It is an enzyme which breaks the H₂ bonds and separates the DNA strands. Thus, a fork is formed at the junction known as replication fork.

B)Single-Stranded Binding (SSB) proteins:These are the protein molecules which attach tightly to the exposed single-stranded DNA in order to stabilize the single-stranded DNA long enough for replication.

C)Primase:It is an enzyme responsible for the synthesis of short RNA primers. AnRNA primer is a small strand of RNA which guides the replication.

D)DNA polymerase:It is an enzyme responsible for catalyzing the synthesis of DNA. It can add nucleotides to the template DNA,

E)Topoisomerase:It is an enzyme responsible for causing nick(cut) in the DNA strand so as to release the tension created during unwinding of DNA.

F)RNAse:It is an enzyme which digests the RNA primers after DNA synthesis is over.

G)DNA ligase:It is an enzyme which seals the gaps in the synthesized DNA strand.

H)Substrates:The four deoxyribonucleoside triphosphates(dNTPs) such as dATPs, dGTPs, dCTPS, and dTTPS are needed as substrates for DNA synthesis. Cleavage of the high-energy bond between phosphates provides the energy for the addition of the nucleotide.

I)Folic acid:It is essential for the synthesis of nitrogen bases.

J)Mg²⁺and Mn²⁺ ions:These are essential for DNA synthesis.

Process of semi-conservative mode of replication of DNA:

The mechanism of DNA replication is a complex and involves many steps. The steps are:

1) Initiation of DNA replication

2) Activation of deoxyribonucleotides

3) Exposure of DNA strands or nitrogen bases and formation of Y-shaped fork

4)Formation of RNA primer

5) Base pairing

6) Formation of new DNA strands

7)Proof-reading and DNA repair

8)Termination of DNA helix formation.

1)Initiation of DNA replication-

The point in the DNA from where replication begins is called initiation point or origin of replication or ori-site. It is formed by the breakage or nicking of phosphodiester bond by the enzyme endonuclease without the removal of nucleotides.

In the prokaryotic cell, DNA forms a single origin of replication and in the eukaryotic cell, DNA has many ori-sites.

2) Activation of deoxyribonucleotides-

Four types of inactive deoxyribonucleoside monophosphates of DNA i:e deAMP, deGMP, deCMP, and deTMP are found in the nucleoplasm react with ATP molecules in the presence of enzymes phosphorylase to form four types of deoxyribonucleotide triphosphates of DNA i:e deATP, deGTP, deCTP, and deTTP. This process is called phosphorylation. [ip=inorganic phosphate]

3)Exposure of DNA strands and formation of replication fork-

Enzyme helicase breaks the hydrogen bonds between the nitrogen bases of two strains of DNA at ori-site. As a result, the two strands of DNA are separated from each other and are in a state of supercoiling tension. To relief from this tension, enzyme topo-isomerase-I or DNA gyrase plays a vital role. The separated strands are established in the Y-shaped structure called replication fork held by single-stranded binding(SSB) protein.

4) Formation of RNA primer-

DNA replication always takes place in 5' to 3' direction on the master or template strands. TO initiate the DNA synthesis, a short sequence of RNA primer is required. RNA primer is synthesized by an enzyme DNA polymerase, called primase. RNA primer is formed on the free end of one DNA strand (3' end) and another in the fork end of another master strand of DNA.

5) Base pairing-

The separated DNA strands are known as templates. The active deoxyribonucleotides present in the nucleoplasm comes to lie opposite to the specific nitrogen bases of both the strand of DNA and bind together according to base pairing rule i:e A=T, G≡C, and T=A, C≡G. With the help of enzyme pyrophosphatase, two extra phosphates present in the deoxyribonucleotide triphosphate separate and energy is released. This energy is used in the formation of hydrogen bonds between the free nucleotides of nucleoplasm and nucleotides of master strands.

deoxyribonucleotide triphosphate (in the presence of pyrophosphatase enzyme) → deoxyribonucleoside monophosphate +2ip

deATP(in presence of pyrophosphatase enzyme)→ deAMP + 2ip + energy

[*ip= inorganic phosphate]

6) Formation of new DNA strands-

DNA polymerase enzyme, energy-rich molecule ATP along with manganese and magnesium ion are responsible for the formation of new DNA strands. New DNA strand is synthesized continuously in 5' to 3' direction on 3' to 5' master strand which is called leading strand.

On the other hand, discontinuous fragments of DNA are also formed in 5' to 3' direction of the other master strand which is called lagging strand. The discontinuous fragments formed by each RNA primer are called Okazaki fragments.

7) Proof-reading and DNA repair-

During replication, the accuracy of base pairing is essential. Sometimes, wrong bases do get in the new strand. The frequency of this introduction of a base is in 100,000. These are noted and removed by the exo-nucleus activity of DNA polymerase-I enzyme (proof-reader). The newly formed DNAstructuree is repaired by DNA ligase enzyme.

8) Termination of DNA helix formation-

Generally, DNA replication stops when two replicating forks meet to each other. But in prokaryotes, a terminating protein called 'Tus' prevents the movement of helicase and indicates the completion of the replication process.

Thus, replication of DNA is a semi-conservative process because parental DNA replicates and forms two daughter DNAs in which one strand of them is parental and another is newly formed.

Reference

Keshari, Arvind K. and Kamal K. Adhikari. A Text Book of Higher Secondary Biology(Class XII). 1st. Kathmandu: Vidyarthi Pustak Bhandar, 2015.

Mehta, Krishna Ram. Principle of biology. 2nd edition. Kathmandu: Asmita, 2068,2069.

Jorden, S.L. principle of biology. 2nd edition . Kathmandu: Asmita book Publication, 2068.2069.