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The autocatalytic function is the capacity of DNA to make a copy of itself. This capacity is known as replication. Watson and Crick proposed a mechanism of replication on the basis of its double helical structure. According to them, the two strands were capable of unwinding and separating and acting as templates during the synthesis of a new nucleotide chain. The method of DNA replication is semi conservative in nature, because the daughter DNA molecule is made of one parental polynucleotide chain and one newly synthesised chain.
According to Watson and Crick, each strand of DNA serves as a template for the synthesis of a new strand which is complementary to the template strand. Due to the specificity of base pairing, each nucleotide of separated chains attract its complementary nucleotide from the nucleoplasm so that A pairs with T and vice versa; C pairs with G and vice versa.
Once, the nucleotides are attached by their hydrogen bonds, the sugar radicals unite through their phosphate components, completing the formation of a new polynucleotide chain. The template and the newly synthesised strand forms a new double strand which is identical to the parent DNA molecule.
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Experimental Evidence of Semiconservative Replication:
The semiconservative replication of DNA was confirmed by an experiment conducted by M Messelson and F W Stahl in 1958. Messelson and Stahl grew E.coli in a medium containing radioactive isotope of nitrogen 15N for many generations. Thus, the entire DNA became labelled with 15N. These cells were transferred to a culture medium containing normal isotope of nitrogen 14N. The cells were left for a specific period of time corresponding to the generation time for E.coli, i.e. the time needed for the cells to divide once and therefore, for the DNA to replicate once.
The samples were subject to density gradient centrifugation. When a solution of cesium chloride (CsCl) is spun in a centrifuge at a very high speed (50 000 revolutions per minute) for many hours, the salt tends to settle down in the centrifuge tube creating a density gradient. In this gradient, the highest ion concentration will be encountered at the bottom. When DNA is mixed with CsCl, it will finally settle at some point in the tube, where the centrifugal force balances the buoyancy of DNA.
The buoyancy of DNA depends on its density, which in turn reflects the ratio of G-C to A-T base pairs. DNA was isolated from the cells at each generation and the DNA molecules containing 15N and 14N was separated on CsCl equilibrium density gradient centrifugation. When the sample is examined under ultraviolet light, the DNA appeared as a narrow band. The positions of the bands of DNA extracted from cells grown in radioactive and normal medium are shown in Fig. 3(a).
It was observed that initially when the entire DNA contained heavy nitrogen, the DNA settled at the bottom. After one generation, the DNA molecule comprised of a hybrid molecule i.e. 14N – 15N, which is made of one strand of heavy DNA and one strand of light DNA. Messelson and Stahl observed that such DNA was actually half dense indicating the presence of hybrid DNA molecules.
After second round of replication there would be four DNA molecules of which two are hybrid in nature i.e. 14N – 15N, with a density as obtained earlier and the remaining two molecules would be made of light strands (14N –14N), which has a density much lighter than the previous two bands. Thus these experiments conclusively demonstrated that DNA replication is semiconservative (Fig. 3b).
