DNA Replication can be defined as a process by which DNA makes a copy of itself. Crick proposed three types of DNA replication method:
- Conservative Method: Parental DNA will remain intact and a new daughter DNA will be formed.
- Dispersive Method: Parental and daughter DNA will mix together and we get a new DNA with different features.
- Semiconservative Method: In this method one strand is parental and another one is daughter strand so there will be 50% parental DNA and 50% daughter DNA.
When studies were carried on later it was found that DNA replicates via Semiconservative model only.
Semiconservative Model Of DNA Replication:
It was proposed by Matthew Meselson and Franklin Stahl using heavy nitrogen (N15) in E.coli
- They grew E.coli in a medium containing N15 ammonium chloride as the only nitrogen source for many generations. N15 is a heavy isotope of nitrogen. N15 was incorporated in newly synthesised DNA as well as other nitrogen-containing compounds. This heavy DNA molecule could be distinguished from normal DNA by centrifugation in a cesium chloride density gradient. A dense solution of CsCl on centrifugation forms density gradient bands of a solution of lower density at the top that increases gradually towards the bottom with the highest density.
- Then they transferred the cells into a medium with normal N14 ammonium chloride and took samples at various definite time intervals as the cells multiplied and extracted the DNA that remained as a double-stranded helix. The various samples were separated independently on CsCl gradients to measure densities of DNA.
- Thus, the DNA that was extracted from the culture after 1st generation i.e just after 20 minutes had a hybrid density. DNA extracted from the culture after another generation was composed of an equal amount of hybrid and light DNA. Increase in the amount of light DNA and decrease in hybrid DNA amount can be possible due to the semiconservative mode of replication.
Note: At every generation parental DNA becomes half.
DNA Replication Mechanism:
The process of replication in living cells requires a set of enzymes. The main is referred to as DNA-dependent DNA polymerase. It can polymerize 2000 base pairs (bp) per second. DNA Replication occurs in the nucleus and during S-phase of cell division.
1.Activation of deoxyribonucleotides:
Four types of deoxyribonucleotides namely dAMP,dGMP, dCMP, dTMP are activated by phosphate, energy and enzyme phosphorylase into triphosphate state. Deoxyribonucleoside serves a dual purpose, in addition to acting as substrates, they provide energy for polymerization reaction because the two terminal phosphates are high energy phosphates.
2. Origin Of Replication:
Replication begins at a particular region of DNA which is called as the origin of replication. Prokaryotes have a small DNA and only one origin of replication. It is called ori-c in E.coli. On the other hand, eukaryotes have large DNA and multiple origins of replication.
3. Unwinding Of Helix:
The unwinding of a double helical parental molecule is brought about by an enzyme helicase which is dependent upon ATP along with magnesium ion. It acts by breaking H-bonds.
The unwinding of DNA molecule into two strands results in the formation of the Y-shaped replication fork. These exposed single strands are stabilised with the help of single-strand binding protein (SSBP). Due to unwinding, supercoiling gets developed on the end of DNA opposite to replication fork. This tension is released by an enzyme topoisomerase. In prokaryotes, DNA Gyrase has topoisomerase as well as helicase activity.
4. Formation Of Primer Strand:
A new strand is now to be synthesised opposite to the parental strands. DNA polymerase III is the true replicase in E.coli but it is incapable of initiating DNA synthesis.
Another enzyme primase synthesizes a short primer strand of RNA. The primer strand then serves as a stepping stone to start errorless replication. Once the synthesis of DNA Replication is completed, this primer RNA strand is removed enzymatically by DNAP-I.
5. Elongation Of New Strand:
The DNA dependent DNA polymerase catalyses polymerization only in one direction that is 5′->3′. Consequently, the replication is continuous on one template strand with polarity 3′->5′. It is known as the leading daughter strand. The replication is discontinuous in the form of Okazaki fragments on other template strands with polarity 5′->3′, this is called lagging daughter strand. The discontinuously synthesised fragments are later joined by the DNA ligase.