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]

fig: Activation of deoxyribonucleotides

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.