DNA Replication: mechanism, enzymes, evidences in favour of semi conservative replication

Introduction:- 
> Replication is the copying of DNA from parental DNA.
> Watson & Crick proposed Semi-conservative model of replication. It suggests that the parental DNA strands act as template for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one new strand.

Evidences in favor of semi conservative replication:- Matthew Messelson & Franklin Stahl (1958) experimentally proved Semi-conservative model.
> They cultured E. coli in a medium containing 15NH4Cl (15N: heavy isotope of N). 15N was incorporated into both strands of bacterial DNA and the DNA became heavier.
> Another preparation containing N salts labeled with 14N is also made. 14N was also incorporated in both strands of DNA and became lighter.
> These 2 types of DNA can be separated by centrifugation in a CsCl density gradient.
> They took E. coli cells from 15N medium and transferred to 14N medium.
> After one generation (i.e. after 20 minutes), they isolated and centrifuged the DNA. Its density was intermediate (hybrid) between 15N DNA and 14N DNA. This shows that the newly formed DNA one strand is old (15N type) and one strand is new (14N type). This confirms semi-conservative replication.
> After II generation (i.e. after 40 minutes), there was equal amounts of hybrid DNA and light DNA.

Enzymes for Replication:- DNA replication is a highly enzyme-dependent process. There are many enzymes involved in DNA replication.
a. DNA polymerase:- It helps in the polymerisation, catalyses and regularises the whole process of DNA replication with the support of other enzymes. dNTPs are the substrate as well as the energy provider for the replication process. 
In prokaryotes, DNA polymerase is of three types:-
i. DNA Polymerase I:- It is responsible for primer extension and proofreading. It is involved in three activities -
5′-3′ polymerase activity
5′-3′ exonuclease activity
3′-5′ exonuclease activity
ii. DNA Polymerase II:- It is a DNA repair enzyme. 
iii. DNA Polymerase III:- It is responsible for in vivo DNA replication.
In eukaryotes, DNA polymerase is of five types:-
b. Helicase:- Helicase is the enzyme, which unzips the DNA strands by breaking the hydrogen bonds between them. Thus, it helps in the formation of the replication fork.
c. Topoisomerase:- All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. The function of topoisomerase is to unwind the chromosomes and DNA double-helix by creating small, reversible cuts in the DNA. Eg.- DNA gyrase in prokaryotes
d. DNA Ligase:- It  is the enzyme which joins together the Okazaki fragments of the discontinuous DNA strands.
e. Primase:- This enzyme helps in the synthesis of RNA primer complementary to the DNA template strand.
f. Endonucleases:- These produce a single-stranded or a double-stranded cut in a DNA molecule.
g. Single-stranded Binding Proteins:- It binds to single-stranded DNA and protects it from forming secondary structures.

Mechanism of DNA:-
> DNA replication starts at a point called origin (ori). In prokaryotes the replication is of single origin while in eukaryotes replication is of multi-origin.
> A unit of replication with one origin is called a replicon.
> During replication, the 2 strands unwind and separate by breaking H-bonds in presence of an enzyme, Helicase. The enzyme gyrase helps with this.
> Unwinding of the DNA molecule at a point forms a ‘Y’-shaped structure called replication fork.
> The separated strands act as templates for the synthesis of new strands.
> DNA replicates in the 5’→3’ direction.
> dNTPs (dATP, dGTP, dCTP & dTTP) act as substrate and also provide energy for polymerization.
> Firstly, a small RNA primer is synthesized in presence of an enzyme, primase.
> In the presence of an enzyme, DNA dependent DNA polymerase, many nucleotides join with one another to primer strand and form a new polynucleotide chain. DNA polymerase-III is the main enzyme in prokaryotes while DNA polymerase ð is main enzyme in eukaryotes.
> The DNA polymerase forms one new strand (leading strand) in a continuous stretch in the 5’→3’ direction (Continuous synthesis).
> The other new strand is formed in small stretches (Okazaki fragments) in 5’→3’ direction (Discontinuous synthesis).
> The Okazaki fragments are then joined together to form a new strand by enzymes, DNA polymerase - I and DNA ligase. This new strand is called lagging strand.
> If a wrong base is introduced in the new strand, DNA polymerase - II can do proof reading.
> E. coli completes replication within 38 minutes. i.e. 2000 bp per second (Genome size = 46 x 106bps). While in eukaryotes the rate of replication is approximately 100 nucleotides per second (Genome size 3.3 x 109bps).
> In eukaryotes, the replication of DNA takes place at S-phase of the cell cycle. Failure in cell division after DNA replication results in polyploidy.