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The copying mechanism to which Watson and Crick referred is called semiconservative and is diagramed in Figure 7-11. The sugar-phosphate backbones are represented by thick ribbons, and the sequence of base pairs is random. Let’s imagine that the double helix is like a zipper that unzips, starting at one end (at the bottom in Figure 7-11). We can see that, if this zipper analogy is valid, the unwinding of the two strands will expose single bases on each strand. Each exposed base has the potential to pair with free nucleotides in solution. Because the DNA structure imposes strict pairing requirements, each exposed base will pair only with its complementary base, A with T and G with C. Thus, each of the two single strands will act as a template, or mold, to direct the assembly of complementary bases to reform a double helix identical to the original. 

The newly added nucleotides are assumed to come from a pool of free nucleotides that must be present in the cell. If this model is correct, then each daughter molecule should contain one parental nucleotide chain and one newly synthesized nucleotide chain. However, a little thought shows that there are at least three different ways in which a parental DNA molecule might be related to the daughter molecules. These hypothetical modes of replication are called semiconservative (the Watson–Crick model), conservative, and dispersive (Figure 7-12). In semiconservative replication, the double helix of each daughter DNA molecule contains one strand from the original DNA molecule and one newly synthesized strand. However, in conservative replication, the parent DNA molecule is conserved, and a single-daughter double helix consists of two newly synthesized strands. 

In dispersive replication, daughter molecules consist of strands each containing segments of both parental DNA and newly synthesized DNA. Meselson–Stahl experiment The first problem in understanding DNA replication was to figure out whether the mechanism of replication was semiconservative, conservative, or dispersive. In 1958, two young scientists, Matthew Meselson and Franklin Stahl, set out to discover which of these possibilities correctly described DNA replication. Their idea was to allow parental DNA molecules containing nucleotides of one density to replicate in a medium containing nucleotides of different densities. If DNA replicated semi-conservatively, the daughter molecules should be half old and half new and therefore of intermediate density. To carry out their experiment, they grew E. coli cells in a medium containing the heavy isotope of nitrogen (15N) rather than the normal light (14N) form.

This isotope was inserted into the nitrogen bases, which then were incorporated into newly synthesized DNA strands. After many cell divisions in 15N, the DNA of the cells was well labeled with the heavy isotope. The cells were then removed from the 15N medium and put into a 14N medium; after one and two cell divisions, samples were taken and the DNA was isolated from each sample. Meselson and Stahl were able to distinguish DNA of different densities because the molecules can be separated from each other by a procedure called cesium chloride gradient centrifugation. If cesium chloride is spun in a centrifuge at tremendously high speeds (50,000 rpm) for many hours, the cesium and chloride ions tend to be pushed by centrifugal force toward the bottom of the tube. Ultimately, a gradient of ions is established in the tube, with the highest ion concentration, or density, at the bottom. 

DNA centrifuged with the cesium chloride forms a band at a position identical to its density in the gradient. DNA of different densities will form bands at different places. Cells initially grown in the heavy isotope 15N showed DNA of high density. This DNA is shown in red on the left-hand side of Figure 7-13a. After growing these cells in the light isotope 14N for one generation, the researchers found that the DNA was of intermediate density, showing half red (15N) and half blue (14N) in the central part. After two generations, both intermediate- and low-density DNA was observed (right-hand side of Figure 7-13a), precisely as predicted by the Watson–Crick model. The replication fork Another prediction of the Watson–Crick model of DNA replication is that a replication zipper, or fork, will be found in the DNA molecule during replication. 

This fork is where the double helix is unwound to produce the two single strands that serve as templates for copying. In 1963, John Cairns tested this prediction by allowing replicating DNA in bacterial cells to incorporate tritiated thymidine ([3H]thymidine)— the thymine nucleotide labeled with a radioactive hydrogen isotope called tritium. Theoretically, each newly synthesized daughter molecule should then contain one radioactive (“hot”) strand (with 3H) and another nonradioactive (“cold”) strand (with 2H). After varying intervals and varying numbers of replication cycles in a “hot” medium, Cairns carefully lysed the bacteria and allowed the cell contents to settle onto a piece of filter paper, which was put on a microscope slide. Finally, Cairns covered the filter with photographic emulsion and exposed it in the dark for 2 months. 

This procedure, called autoradiography, allowed Cairns to develop a picture of the location of 3H in the cell material. As 3H decays, it emits a beta particle (an energetic electron). The photographic emulsion detects a chemical reaction wherever a beta particle strikes the emulsion. The emulsion can then be developed like a photographic print so that the emission track of the beta particle appears as a black spot or grain. After one replication cycle in [3H]thymidine, a ring of dots appeared in the autoradiograph. Cairns interpreted this ring as a newly formed radioactive strand in a circular daughter DNA molecule, as shown in Figure 7-14a. It is thus apparent that the bacterial chromosome is circular—a fact that also emerged from the genetic analysis described earlier (Chapter 5). In the second replication cycle, the forks predicted by the model were indeed seen. 

Furthermore, the density of grains in the three segments was such that the interpretation shown in Figure 7-14b could be made: the thick curve of dots cutting through the interior of the circle of DNA would be the newly synthesized daughter strand, this time consisting of two radioactive strands. Cairns saw all sizes of these moon-shaped, autoradiographic patterns, corresponding to the progressive movement of the replication forks