DNA replication:

Duplication of one parental strand of DNA to form new daughter strand by following the law of semiconservativeness. And the newly synthesized double helix DNA has one parental and one new strand.

Experimental proof for semi-conservative mode of DNA replication

Matthew Meselson and Franklin Stahl performed experiments on E. coli to prove that DNA replication is semi-conservative.

1. They grew E. coli in a medium containing ¹⁵NH₄Cl (in which ¹⁵N is the heavy isotope of nitrogen) for many generations.
2. As a result, ¹⁵N not incorporated into newly synthesized DNA.
3. This heavy DNA can be differentiated from normal DNA by centrifugation in caesium chloride (CsCl) density gradient.
4. Then they transferred the cells into a medium with normal ¹⁴NH₄Cl and took the samples at various definite time intervals as the cells multiplied.
5. The extracted DNAs were centrifuged and measured to get their densities.
6. The DNA extracted from the culture after one generation of transfer from the ¹⁵N medium to ¹⁴N medium (i.e., after 20 minutes; E. coli divides every 20 minutes) showed an intermediate hybrid density.
7. The DNA extracted from culture after two generations (i.e., after 40 minutes) showed equal amounts of light DNA and hybrid DNA.
8. Similar experiment was performed by Taylor and colleagues in 1958, on Vicia faba to prove that the DNA in chromosome also replicate semi-conservatively.

Enzymes for DNA replication

DNA-dependent DNA polymerase: It catalyses the polymerization of deoxynucleotides on DNA template.

Helicase: It unwinds the DNA strand to form the replication fork.

DNA ligase: It joints the Okazaki fragments which are formed on the lagging strand.

 Process of DNA Replication

DNA replication beings at a unique and fixed point called origin of replication or ‘ori’.

Initiation

1) The complementary strands of DNA double helix are separated by two enzymes, DNA gyrase and DNA helicase. This is called unwinding of double-stranded DNA.

2) The separated strands tend to rewind. Unwinding of double-stranded DNA forms a Y-shaped configuration in the DNA duplex, which is called replication fork.

Elongation

3) An enzyme called primase initiates replication of the strand oriented in the 3’ (towards origin) ⟶ 5’.

4) DNA polymerase progressively adds deoxyribonucleotides to the free 3’ –end of the growing polynucleotide chain so that replication of the 3’ ⟶ 5’ strand of the DNA molecule is continuous.

5) The replication of 3’ ⟶ 5’ strand is continuous and it is called leading strand, while the replication of second strand (5’ ⟶ 3’ strand) of the DNA molecules is discontinuous and it is known as the lagging strand.

6) The replication of lagging strand generates small polynucleotide fragments called ‘Okazaki fragments.”These Okazaki fragments are then joined together by enzyme called DNA ligase.