Semi conservative mode of D.N.A. replication was first proposed by Watson & Crick. Later on it was experimentally proved by Meselson. & Stahl (1958) on E- Coli and Taylor on Vi cia faba (1958).To prove this method, Taylor used Radiotracer Technique in which Radioisotopes (tritiated thymidine = `text()_(1)H^3`) were used. Meselson and Stahl used heavy isotope (N 1'') and Cairns (1963) used radioactive thymidine

`->` Due to the replication of active Thymidine containing D.N.A., two D.N.A. molecules were obtained in `50%` radioactivity was found.

`->` When these two D.N.A. molecules containing active Thymidine were made to replicate, the next time four D.N.A. molecules were obtained. Out of these 4 D.N.A. . 2 D.N.A. molecules were radioactive and remaining 2 were not radioactive.

`->` In the same sequence, the obtained D.N.A. molecules were further made to replicate then also the no. : of radioactive D.N.A. remains 2..

The following steps are included in D.N.A. replication ,.

`->` The separation of `2` chains of D.N.A is termed as unzipping. And it takes place due to thr breaking of H-bonds. The process of unzipping starts at a certain specific point which is termed as initiation point or origin of replication. In prokaryotes there occurs only one origin of replication but in eukaryotes there occur many origin of replication i.e. unzipping starts at many points simultaneously. At the place of orogin the topoisomerase enzyme (a type of endonuclease) induces a cut in one strand of DNA (Nicking) to relax the two strands of DNA.

`->` The enzyme responsible for unzipping (t:reaking the hydrogen tx)nds) is Helicase {= Swivelase). In the proces..s of
unzipping `Mg^(+2)` act as cofactor. Unzipping takes place in alkaline medium.

`->` A protein, "Helix destabilizing protein" or SSB (single stranded DNA binding protein) prevents the formation
of bends or loops in separated strands.

DNA-Gyrase - A type of topoisomerase prevents supercoiling of ON/\.

Note:-
The process of D.N.A. replication takes a few minutes in prokaryotes and a few hours in Eukaryotes.

`->` To start the synthesis of new chain, special type of R.N.A. is required which is termed as R.N.A. Primer. The formation of R.N.A. primer is catalysed by an enzyme - R.N.A. Polymerase (primase). Synthesis of RNA-primer takes place in `5' ->- 3'` direction. After the formation of new chain, this R.N.A. is removed. For the formation of new chain Nucleotides are obtained from Nuceloplasm. In the nucleoplasm, Nucleotides are present in the form of triphosphaies like dATP, dGTP, dCTP, dTn) etc.

`->` During replication, the `2` phosphate groups of all Nucleotides are separated. In this process energy is yeilded which is consumed in D.N.A. replication. So, it is clear that D.N.A. does not depend on mitochondria for it's energy requirements.

`->` Energetically replication is a very expensive process .. Daoxyribonucleoside triphosphase serve duas purposes in addition to acting as substrates they provide energy for polymerisation.

`->` The formation of new chain always takes place in 5' - 3' direction. 1\s a result of this, one chain of D.N.A. is continuously formed and it is termed as Leading strand. The formation of second chain begins from the centre and not from the terminal points, so this chain is discontinuous and is made up of small segments called Okazaki Fragments. This discontinuous chain is termed as Lagging strand. Ultimately all these segments joined together and a complete new chain is formed.

`->` The Okazaki segments are joined together by an enzyme DNA LiBase.(Khorana).

`->` The formation of new chains is catalysed by an enzyme DNA Polymerase. In prokaryotes it is of 3 types.

(1) DNA- Polymerase I :-This was discovered by KORNBERG (1957). So it is also called as 'kornberg's enzyme'. Kornberg also synthesized DNA first of all, in the laboratory. This enzyme functions as exonuclease. It separates RNA - primer from DNA and also fills the gap.lt is also known as DNA-repair enzyme.

(2) DNA - Polymerase II :- It is least reactive in replication process. It is also helpful in DNA-repairing in absence of DNA-polymerase-! and DNA polymerase-III

(3) DNA - Polymerase Ill :- This is the main enzyme in DNA - Replication. It is most important , It was discovered by Delucia and Cairns. The larger chains are formed by this enzyme. This is also known as Replicase. DNA -polymerase III is a complex enzyme composed of seven polypeptides

i.e. `alpha , sigma , theta , beta , gamma_1 , delta , gamma_2`


In Eucaryotes, there occur five types of DNA-polymerase enzyme.

(1) `alpha`-DNA - polymerase = 11 is concern with lagging strand synthesis.

(2) `beta`-DNA - polymerase = It concerned with DNA repair.

(3) `gamma`-DNA - polymerase = It concerned with replication of cytoplasmic DNA.

(4) `delta` -DNA - polymerase l1 is concern with leading strand synthesis.

(5) `sigma`- DNA polymerase = It is concern with proof reading.

`->` Thus DNA - Replication process is completed with the effect of different enzymes.

`->` In the semi conservative mode of replication each daughter DNA molecule receives one chain of polynucleotides
from the mother DNA - molecule and the second chain is synthesized.

`->` All DNA polymerase `I, II` and `Ill` enzymes have `5'-3"'` polymerisation activity and `3'-5"'` exonuclease activity.
`->` Any failure in cell division after DNA replication result into polyploidy.
`->` Difference between DNAs and DNase is that DNAs menas many DNA and DNase means DNA digestive enzymes.