DNA Replication - Part 1

DNA Replication is the biological process of producing two identical replicas of DNA from the original DNA molecule. DNA replication occurs in all living organisms and acts as the most essential part for the biological inheritance. The cell possesses the distinctive property of division, which makes replication of DNA essential. It occurs in the S-sub stage of inter phase of the cell cycle. Replication in prokaryotes is much better understood than in the eukaryotes. The basic requirements and components of replication are the same in both prokaryotes and eukaryotes.

Methods of Replication

The three possible methods of the replication of DNA are:

1. Conservative replication

DNA replication results in one molecule that consists of both original strands and another molecule that consists of two new strands.

2. Semi-conservative replication

The two strands of the DNA unwind from each other, and each strand acts as a template for the synthesis of a new complementary strand. This results in two DNA molecules with one original strand and one new strand. This is the most accepted model of DNA replication and was described by Watson and Crick.

3. Dispersive replication

DNA replication results in two DNA molecules that are mixtures of parental and new DNA. In this model,each individual strand is a patchwork of the original and new DNA.

The experimental proof of semi conservative model of DNA replication

It is now proven that DNA replicates semi conservatively. It was shown first inn Escherichia coli and subsequently in higher organisms, such as plants and human cells. Matthew Meselson and Franklin Stahl performed their experiment and proved it in 1958.

Fig: Experiment of Messelson and Stahl

In their experiment, the DNA was labelled with heavy nitrogen (N¹⁵) and was then allowed to replicate in a medium containing N¹⁴. If the replication is semi conservative, then after first generation of replication, one of the two strands would have N¹⁵ and the other strand would have normal nitrogen N¹⁴. The result would be that this molecule would have a density which is intermediate between N¹⁵ DNA and N¹⁴ DNA. This density will gradually fall and in the following generations of replication, the density would approach that of N¹⁴ DNA.

Basic Requirements for DNA Replication

The mechanism of DNA replication is complex and involves many steps. Several enzymes, protein factors and metal ions are required for this process.

1. DNA helicase

It is the enzyme which breaks the hydrogen bonds and separates the DNA strands. Thus, a fork is formed at the junction known as the replication fork.

2. Single Stranded Binding (SSB) proteins

These are the protein molecules which attach tightly to the exposed single stranded DNA in order to stabilize the single stranded DNA long enough for replication.

3. Primase

It is an enzyme responsible for the synthesis of short RNA primers. A RNA primer is a small strand of RNA which guides the replication.

4. DNA polymerase

It is an enzyme responsible for catalyzing the synthesis of DNA. It can add nucleotide in the template DNA.

5. Topoisomerase

It is an enzyme responsible for causing nick in the DNA strand so as to release the tension created during unwinding of DNA. Nick refers to cutting or breaking of phospho-diester bond.

6.RNAse

It is an enzyme which digests the RNA primers after DNA synthesis is over.

7. DNA ligase

It is an enzyme which seals the gaps in the synthesized DNA strand.

8. Substrates

The four deoxyribonucleoside triphosphates (dNTPs) such as dATPs, dGTPs, dCTPs, dTTPs are needed as substrates for DNA synthesis. Cleavage of the high-energy bond between the phosphates provides the energy for the addition of the nucleotide.

9. Folic acid

It is essential for the synthesis of nitrogen bases.

10. Mg²⁺ and Mn²⁺ ions

These are essential for DNA synthesis.