The double-helical model of DNA and the presence of specific base pairs immediately suggested how the genetic material might replicate. The sequence of bases of one strand of the double helix precisely determines the sequence of the other strand: a guanine base on one strand is always paired with a cytosine base on the other strand, and so on. Thus, separation of a double helix into its two component strands would yield two single-stranded templates onto which new double helices could be constructed, each of which would have the same sequence of bases as the parent double helix. Consequently, as DNA is replicated, one of the strands of each daughter DNA molecule is newly synthesized, whereas the other is passed unchanged from the parent DNA molecule. This distribution of parental atoms is achieved by semiconservative replication.

115

Matthew Meselson and Franklin Stahl carried out a critical test of this hypothesis in 1958. They labeled the parent DNA with ¹⁵N, a heavy isotope of nitrogen, to make it denser than ordinary DNA. The labeled DNA was generated by growing E. coli for many generations in a medium that contained ¹⁵NH₄Cl as the sole nitrogen source. After the incorporation of heavy nitrogen was complete, the bacteria were abruptly transferred to a medium that contained ¹⁴N, the ordinary isotope of nitrogen. The question asked was: What is the distribution of ¹⁴N and ¹⁵N in the DNA molecules after successive rounds of replication?

The distribution of ¹⁴N and ¹⁵N was revealed by the technique of density-gradient equilibrium sedimentation. A small amount of DNA was dissolved in a concentrated solution of cesium chloride having a density close to that of the DNA (1.7 g cm⁻³). This solution was centrifuged until it was nearly at equilibrium. At that point, the opposing processes of sedimentation and diffusion created a gradient in the concentration of cesium chloride across the centrifuge cell. The result was a stable density gradient ranging from 1.66 to 1.76 g cm⁻³. The DNA molecules in this density gradient were driven by centrifugal force into the region where the solution’s density was equal to their own. The DNA yielded a narrow band that was detected by its absorption of ultraviolet light. A mixture of ¹⁴N DNA and ¹⁵N DNA molecules gave clearly separate bands because they differ in density by about 1% (Figure 4.20).

DNA was extracted from the bacteria at various times after they were transferred from a ¹⁵N to a ¹⁴N medium. Analysis of these samples by the density-gradient technique showed that there was a single band of DNA after one generation. The density of this band was precisely halfway between the densities of the ¹⁴N DNA and ¹⁵N DNA bands (Figure 4.21).

116

The absence of ¹⁵N DNA indicated that parental DNA was not preserved as an intact unit after replication. The absence of ¹⁴N DNA indicated that all the daughter DNA derived some of their atoms from the parent DNA. This proportion had to be half because the density of the hybrid DNA band was halfway between the densities of the ¹⁴N DNA and ¹⁵N DNA bands.

After two generations, there were equal amounts of two bands of DNA. One was hybrid DNA, and the other was ¹⁴N DNA. Meselson and Stahl concluded from these incisive experiments that replication was semiconservative, and so each new double helix contains a parent strand and a newly synthesized strand. Their results agreed perfectly with the Watson–Crick model for DNA replication (Figure 4.22).

During DNA replication and transcription, the two strands of the double helix must be separated from each other, at least in a local region. The two strands of a DNA helix readily come apart when the hydrogen bonds between base pairs are disrupted. In the laboratory, the double helix can be disrupted by heating a solution of DNA or by adding acid or alkali to ionize its bases. The dissociation of the double helix is called melting because it occurs abruptly at a certain temperature. The melting temperature (T_m) of DNA is defined as the temperature at which half the helical structure is lost. Inside cells, however, the double helix is not melted by the addition of heat. Instead, proteins called helicases use chemical energy (from ATP) to disrupt the helix (Chapter 28).

Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases, an effect called hypochromism. Thus, the melting of nucleic acids is readily monitored by measuring their absorption of light, which is maximal at a wavelength of 260 nm (Figure 4.23).

Separated complementary strands of nucleic acids spontaneously reassociate to form a double helix when the temperature is lowered below T_m. This renaturation process is sometimes called annealing. The facility with which double helices can be melted and then reassociated is crucial for the biological functions of nucleic acids.

117

The ability to melt and reanneal DNA reversibly in the laboratory provides a powerful tool for investigating sequence similarity. For instance, DNA molecules from two different organisms can be melted and allowed to reanneal, or hybridize, in the presence of each other. If the sequences are similar, hybrid DNA duplexes, with DNA from each organism contributing a strand of the double helix, can form. The degree of hybridization is an indication of the relatedness of the genomes and hence the organisms. Similar hybridization experiments with RNA and DNA can locate genes in a cell’s DNA that correspond to a particular RNA. We will return to this important technique in Chapter 5.