Watson and Crick recognized that their DNA model suggests a replication mechanism in which the two parental strands separate, allowing each separated strand to serve as a template for the synthesis of a complementary strand. According to this replication mechanism, which is termed the semiconservative model for DNA replication, each double-stranded daughter DNA molecule will have a conserved DNA strand that is derived from the parental DNA and a newly synthesized strand. At the time the semiconservative model was proposed, DNA denaturation was not understood and strand separation was, for a variety of reasons, considered to be impossible. Therefore, two alternative models, the conservative and dispersive models, also seemed possible.
The conservative model of replication makes two assumptions. First, the two strands of the double helix unwind at the replication site only to the extent needed for the base sequence there to be read by the polymerizing enzyme. Second, the two original strands remain entwined after replication so that one of the two DNA molecules present after replication contains both original strands (is conserved) and the other DNA molecule is made of two new strands.
The dispersive model of replication shares some of the features of the conservative model but predicts that each strand of the daughter DNA molecules has interspersed sections of both old and new DNA. Comparison of the three mechanisms reveals that conservative replication make different predictions about the composition of daughter DNA molecules after one or two rounds of replication. Therefore, it would be possible to establish the correct model, if some method could be devised to distinguish between new and old DNA strands.
In 1958, just five years after the Watson-Crick Model was proposed, Matthew Meselson and Franklin Stahl realized that they might be able to use equilibrium density gradient centrifugation to demonstrate that DNA replication is semiconservative. They started by culturing E. coli for many generations in a growth medium containing NH4 Cl as the sole source of nitrogen so that the purine and pyrimidine bases in DNA were uniformly labeled with the heavy isotope. Then they transferred the bacteria to a new growth medium containing [14N] NH4 Cl, removed samples from the culture at various times, extracted DNA from the samples, added the extracted DNA to a concentrated cesium chloride solution, and subjected the mixture to a high-speed centrifugation for about one day to establish a cesium chloride equilibrium density gradient (see Chapter 5). Photographs of the gradient solutions were taken using ultraviolet light with a wavelength of 260 nm to reveal dark DNA bands. The results of the experiment are shown in. The column labeled generations indicates the number of generations that the cells were incubated in the growth medium containing NH4Cl. The photographs are oriented so that cesium chloride density increases from left to right (top to bottom of the centrifuge tube). The tracing to the right of each photograph shows the 260 nm light absorption of the DNA in the photograph.
