The Watson–Crick model for DNA replication (introduced in Chapter 2) assumed that as new strands of DNA are made, they follow the usual base-pairing rules of A with T and G with C. The model also proposed that the two parental strands separate and that each then serves as a template for a new progeny strand. This is called semiconservative replication because each daughter duplex has one parental strand and one new strand (Figure 20.1a). In other words, one of the parental strands is “conserved” in each daughter duplex. However, this is not the only possibility. Another potential mechanism (Figure 20.1b) is conservative replication, in which the two parental strands stay together and somehow produce another daughter helix with two completely new strands. Yet another possibility is random dispersive replication, in which the DNA becomes fragmented so that new and old DNAs coexist in the same strand after replication (Figure 20.1 c).
In 1958, Matthew Meselson and Franklin Stahl performed a classic experiment to distinguish among these three possibilities. They labeled E. coli DNA with heavy nitrogen (15N) by growing cells in a medium enriched in this nitrogen isotope. This made the DNA denser than normal. Then they switched the cells to an ordinary medium containing primarily 14N, for various lengths of time. Finally, they subjected the DNA to CsCl gradient ultracentrifugation to determine the density of the DNA. Figure 20.2 depicts the results of a control experiment that shows that 15N- and 14N-DNAs are clearly separated by this method.
What outcomes would we expect after one round of replication according to the three different mechanisms? If replication is conservative, the two dense parental strands
will stay together, and another, newly made DNA duplex will appear. Because this second duplex will be made in the presence of light nitrogen, both its strands will be less
dense. The dense/dense (D/D) parental duplex and less dense/less dense (L/L) progeny duplex will separate readily in the CsCl gradient (Figure 20.3a). On the other hand, if replication is semiconservative, the two dense parental strands will separate and each will be supplied with a new, less dense partner. These D/L hybrid duplexes will have a density halfway between the D/D parental duplexes and L/L ordinary DNA (Figure 20.3b). Figure 20.4 shows that this is exactly what happened; after the first DNA doubling, a single band appeared midway between the labeled D/D DNA and a normal L/L DNA. This ruled out conservative replication, but was still consistent with either semiconservative or random dispersive replication.
Everything i do, i do it for you