In this article we will discuss about:- 1. Experimental Proof of Replication of DNA 2. Enzymes for DNA Replication 3. Process of Replication of DNA.
In addition to the double helical structure of DNA, Watson and Crick also proposed a scheme for DNA replication. According to this scheme, the two strands of double-helix would separate and act as a template for the synthesis of new complementary strands.
After the completion of replication, each DNA molecule has one parental and one newly synthesised strand.
This scheme for DNA replication was known as semi-conservative DNA replication.
Experimental Proof of Replication of DNA:
Matthew Meselson and Franklin Stahl conducted an experiment with Escherichia coli (in 1958).
(i) They grew many generations of E. coli in a medium that contained 15NH4Cl (15N is the heavy isotope of nitrogen.) as the only source of nitrogen. The result was that N was incorporated into the newly synthesised DNA.
By centrifugation in a Cesium Chloride (CsCl) density gradient this heavy DNA molecule could be distinguished from the normal DNA.
(ii) These cells were then transferred into a medium containing normal 14NH4Cl.
(iii) At definite time intervals, as the cells multiplied, samples of the growth were taken; and the DNA which remained as double-stranded helices were extracted.
(iv) These samples were separated independently on CsCl gradient to measure the densities of DNA.
(v) The DNA obtained from the sample of the growth of one generation after the transfer from 15N to 14N medium, had a hybrid or intermediate density.
(vi) While the DNA obtained from sample growth after another generation (II generation), consisted equal amount of hybrid DNA and light DNA.
(vii) Similar experiments on Vicia fabd (faba beans) were conducted by Taylor and Colleagues in 1958. The results were the same, i.e., DNA replicates semi-conservatively.
Enzymes for DNA Replication:
The process of replication requires following sets of catalysts (enzyme).
These are given below:
1. DNA-dependent DNA Polymerase:
It is the main enzymes which uses a DNA template to catalyse the polymerisation of deoxynucleotides.
The average rate of polymerisation is 2000 bp (base pairs) per second approximately.
These enzymes are highly efficient as they have to catalyse polymerisation of large number of nucleotide in a very short period of time.
These polymerases need to catalyse the replication process with high degree of accuracy, otherwise any mistake would result in mutations.
2. Helicase:
It unwinds the DNA strand for the formation of a replication fork.
3. DNA Ligase:
It facilitates the joining of DA strands together by catalysing the formation of phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA.
In a long DNA molecule, replication takes place within a small opening of the DNA helix, known as replication fork.
DNA dependent DNA polymerases catalyse polymerisation only in one direction (5′ → 3′). This brings additional complications at the replication fork.
On the 5′ strand, replication is continuous, while on the 5′ → 3’strand, replication is discontinuous. Thus, the fragments synthesised by discontinuous replication are later joined by the enzyme DNA ligase.
Process of Replication of DNA:
Replication is an energy expensive process, deoxyribonucleoside triphosphate serve the dual purpose of:
(i) Acting as a substrate.
(ii) Providing energy (from two terminal phosphates). DNA replication begins at unique and fixed position in a DNA molecule known as origin of replication ori.
These are definite regions in DNA, where the replication originates.
Due to the requirement of the origin of replication, if a piece of DNA needs to be propagated during recombination, a vector is required. The vectors provide the origin of replication.
Note:
In eukaryotes, the replication of DNA takes place at S-phase of the cell cycle. After DNA replication, if the cell division fails to take place, it results in polyploidy.
