DNA Replication

DNA Replication

     Watson and Crick structure of DNA itself suggests for replica-tion process. Strong experimental support suggest that the mode of replication is semi conservative replication. That means half of the DNA is conserved at the molecular level. Each strand of the DNA double helix can serve as a template for the synthesis of its complementary strand. (Figure 35.9)

       Each replicative DNA molecule will consist of one old and one new strand. Hence the process is known as semi conservative replica-tion. In 1958, Mathew Meselson and Franklin Stahl gave experimental evidence for semi conservative process that is being used by bacterial cells. They grew E.coli cells for many generations in medium contain-ing heavy isotope of nitrogen source 15N. After many generations all nitrogen containing molecules in E.coli cells including nitrogen bases of DNA contained 15N. These cell were then transferred to a medium containing only 14N (light isotope). All the subsequent DNA synthesis, during replication contained 14N.

Cell samples were removed at periodic time intervals from the growth medium and from each sample DNA was isolated and sub-jected to sedimentation equilibrium centrifugation.

     The heavy isotope 15N containing DNA will reach equilibrium in a gradient point closer to the bottom of the tube whereas the 14N con-taining DNA occupy a higher position in the tube as equilibrium. The density of the DNA is determined by using ultra centrifugation on a CsCl gradient. CsCl forms are density gradient with the greatest den-sity at the bottom.

After first generation the isolated DNA occupies an intermediate density suggesting each replicated molecule is composed of one new14N strand and one old 15N.

     After second generation, the two density bands were observed,

     After second generation, the two density bands were observed, one at intermediate and one at lighter, which corresponds to the 14N position in the gradient.

       This experimental results and other experiments repeated by Meselson and Stahl with prokaryotes suggested that semi conservative mechanism of replication is universal.