DNA Replication: DNA replication is the process by which the genome’s DNA is copied in cells. Before a cell divides, it must first copy (or replicate) its entire genome so that each resulting daughter cell ends up with its own complete genome.

DNA Replication

• Watson and Crick suggested that the replication of DNA is semiconservative.
• Meselson and Stahl in 1958 experimentally proved that the DNA replicates semi conservatively.
• Taylor et al in another experiment on fava beans (Vicia faba) using radioactive thymidine proved that the replication on DNA is semiconservative.
• Enzyme DNA polymerase catalyses DNA replication. It can polymerise only in 5’→3’ direction.
• Replication is initiated at the origin of replication.
• Deoxyribonucleoside triphosphate provides energy for the polymerisation reaction and also acts as a substrate.
• A small part of DNA opens up making a replication fork, where replication occurs.
• Replication is continuous in a strand with 5’→3’ direction, called leading strand, where the template strand has 3’→5’ polarity, called leading strand template.
• Replication is discontinuous in the other strand, where the template strand has 5’→3’ polarity, called lagging strand template.
• The discontinuous fragments, called Okazaki fragments are joined together by the enzyme DNA ligase.
• In eukaryotic cells, the replication takes place during s-phase of the cell cycle.
• If cell division doesn’t occur after the replication, it results in polyploidy of chromosomes.

Experimental proof that DNA Replication is semi-conservative

• E. coli was grown in a medium containing 15NH4C1 as the only nitrogen source for many generations. 15N got incorporated into newly synthesised DNA (and other nitrogen containing compounds). This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
• They then transferred the cells into a medium with normal 14NH4Cl and took samples at various definite intervals as the cells multiplied and extracted the DNA that remained as double stranded helices. DNA samples were separated independently on CsCl gradients to measure DNA densities.
• The DNA that was extracted from the culture, one generation (after 20 min) after the transfer from 15 N to 14N medium had a hybrid or intermediate density. DNA extracted from the culture after another generation (after 40 min) was composed of equal amounts of this hybrid DNA and of light DNA.
• Very similar experiments were carried out by Taylor and Colleagues on Vicia faba (faba beans) using radioactive thymidine and the same results, i.e. DNA replicates semiconservatively, were obtained as in earlier experiments.

DNA Replication Machinery and Enzymes

• DNA replication machinery and enzymes process of replication requires a set of catalysts (enzymes).
• The main enzyme is DNA-dependent DNA polymerase, since it uses a DNA template to catalyse the polymerisation of deoxynucleotides. The average rate of polymerisation by these enzymes is approximately 2000 bp/second.
• These polymerases has to catalyse the reaction with high degree of accuracy because any mistake during replication would result into mutations.
• DNA polymerisation is an energy demanding process, so deoxyribonucleoside triphosphates serve dual purposes, i.e. act as substrates and provide energy for polymerisation reaction.
• Many additional enzymes are also required in addition to DNA-dependent DNA polymerase.
• Replication in DNA strand occurs within a small opening of the DNA helix, known as replication fork.
• DNA-dependent DNA polymerases catalyse polymerisation only in one direction, i.e. 5′ -> 3. It creates additional complications at the replicating fork. Consequently, on one strand (template 3′-5′), the replication is continuous, while on the other strand (template 5′-3′), it is discontinuous. The discontinuously synthesised fragments called Okazaki fragments are later joined by DNA ligase.
• DNA polymerases cannot initiate the process of replication on their own. Also, replication does not initiate randomly at any place in DNA. So, there is a definite region in E.coli DNA where the replication originates. The region is termed as origin of replication.
• Due to this requirement, a piece of DNA, if needed to be propagated during recombinant DNA procedures, requires a vector. The vectors provide the origin of replication.

Follow on Facebook

By Team Learning Mantras