Semiconservative replication

Figure 8-10 diagrams the possible basic mechanism for DNA replication proposed by Watson and Crick. The sugar-phosphate backbones are represented by lines, 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 8-10). We can see that, if this zipper analogy is valid, the unwinding of the two strands will expose single bases on each strand. Because the pairing requirements imposed by the DNA structure are strict, each exposed base will pair only with its complementary base. Because of this base complementarity, each of the two single strands will act as a template, or mold, and will begin to reform a double helix identical with the one from which it was unzipped. The newly added nucleotides are assumed to come from a pool of free nucleotides that must be present in the cell.

Figure 8-10

The model of DNA replication proposed by Watson and Crick is based on the hydrogen-bonded specificity of the base pairs. Complementary strands are shown in different colors. This drawing is a simplified version of the current picture of replication but (more...)

If this model is correct, then each daughter molecule should contain one parental nucleotide chain and one newly synthesized nucleotide chain. This prediction has been tested in both prokaryotes and eukaryotes. 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 are called semicon-servative (the Watson-Crick model), conservative, and dispersive (Figure 8-11). In semiconservative replication, each daughter duplex contains one parental and one newly synthesized strand. However, in conservative replication, one daughter duplex consists of two newly synthesized strands, and the parent duplex is conserved. Dispersive replication results in daughter duplexes that consist of strands containing only segments of parental DNA and newly synthesized DNA.

Figure 8-11

Three alternative patterns for DNA replication. The Watson-Crick model would produce the first (semiconservative) pattern. Light blue lines represent the newly synthesized strands.

Meselson-Stahl experiment

In 1958, Matthew Meselson and Franklin Stahl set out to distinguish among these possibilities. 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 were 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. DNA was extracted from the cells in each of these samples and put into a solution of cesium chloride (CsCl) in an ultracentrifuge.

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 Cs⁺ and Cl⁻ ions is established in the tube, with the highest ion concentration at the bottom. Molecules of DNA in the solution also are pushed toward the bottom by centrifugal force. But, as they travel down the tube, they encounter the increasing salt concentration, which tends to push them back up owing to the buoyancy of DNA (its tendency to float). Thus, the DNA finally “settles” at some point in the tube where the centrifugal forces just balance the buoyancy of the molecules in the cesium chloride gradient. The buoyancy of DNA depends on its density (which in turn depends on the ratio of G–C to A–T base pairs). The presence of the heavier isotope of nitrogen changes the buoyant density of DNA. The DNA extracted from cells grown for several generations on ¹⁵N medium can be readily distinguished from the DNA of cells grown on ¹⁴N medium by the equilibrium position reached in a cesium chloride gradient. Such samples are commonly called heavy and light DNA, respectively.

Meselson and Stahl found that, one generation after the heavy cells were moved to ¹⁴N medium, the DNA formed a single band of an intermediate density between the densities of the heavy and light controls. After two generations in ¹⁴N medium, the DNA formed two bands: one at the intermediate position, the other at the light position (Figure 8-12). This result would be expected from the semiconservative mode of replication; in fact, the result is compatible with only this mode if the experiment begins with chromosomes composed of individual double helices (Figure 8-13).

Figure 8-12

Centrifugation of DNA in a cesium chloride (CsCl) gradient. Cultures grown for many generations in ¹⁵N and ¹⁴N media provide control positions for heavy and light DNA bands, respectively. When the cells grown in ¹⁵N are transferred to a ¹⁴N medium, the (more...)

Figure 8-13

Only the semiconservative model of DNA replication predicts results like those shown in Figure 8-12: a single intermediate band in the first generation and one intermediate and one light band in the second generation. (See Figure 8-11 for explanation (more...)

Autoradiography

The Meselson-Stahl experiment on E. coli was essentially duplicated in 1958 by Herbert Taylor on the chromosomes of bean root-tip cells, by using a cytological technique. Taylor put root cells into a solution containing tritiated thymidine ([³H]thymidine)—the thymine nucleotide labeled with a radioactive hydrogen isotope called tritium. He allowed the cells to undergo mitosis in this solution so that the [³H]thymidine could be incorporated into DNA. He then washed the tips and transferred them to a solution containing nonradioactive thymidine. Addition of colchicine to such a preparation inhibits the spindle apparatus so that chromosomes in metaphase fail to separate and sister chromatids remain “tied together” by the centromere.

The cellular location of ³H can be determined by autoradiography. As ³H decays, it emits a beta particle (an energetic electron). If a layer of photographic emulsion is spread over a cell that contains ³H, a chemical reaction takes place 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. The cell can also be stained, making the structure of the cell visible, to identify the location of the radioactivity. In effect, autoradiography is a process in which radioactive cell structures “take their own pictures.”

Figure 8-14 shows the results observed when colchicine is added during the division in [³H]thymidine or during the subsequent mitotic division. It is possible to interpret these results by representing each chromatid as a single DNA molecule that replicates semiconservatively (Figure 8-15).

Figure 8-14

Diagrammatic representation of the autoradiography of chromosomes from cells grown for one cell division in the presence of the radioactive hydrogen isotope ³H (tritium) and then grown in a nonradioactive medium for a second mitotic division. Each dot represents (more...)

Figure 8-15

An explanation of Figure 8-14 at the DNA level. Light blue lines represent radioactive strands. In the second replication (which takes place in nontritiated solution), both the ³H strand and the nontritiated strand incorporate nonradioactive nucleotides, (more...)

Harlequin chromosomes

With the use of a more modern staining technique, it is now possible to visualize the semiconservative replication of chromosomes at mitosis without the aid of autoradiography. In this procedure, the chromosomes are allowed to go through two rounds of replication in bromodeoxyuridine (BUdR). The BUdR labeling pattern, shown in Figure 8-16a, is the reciprocal of that of Figure 8-15, because the BUdR is used in both replications, rather than being replaced by normal thymidine for the second replication as in the autoradiographic process. The chromosomes are then stained with fluorescent dye and Giemsa stain; this process distinguishes hybrid chromatids with one BUdR-containing strand and one original strand (dark stain) from those in which both strands contain BUdR (light stain), and generates so-called harlequin chromosomes (Figure 8-16b). (Note, in passing, that harlequin chromosomes are particularly favorable for the detection of sister-chromatid exchange at mitosis; two examples are seen in Figure 8-16b). Using similar techniques. Taylor showed that chromosome replication at meiosis also is semiconservative.

Figure 8-16

(a) A diagrammatic representation of the production of harlequin chromosomes. In this procedure, the chromosomes go through two rounds of replication in the presence of bromodeoxyuridine (BUdR), which replaces thymidine in the newly synthesized DNA. The chromosomes (more...)

Chromosome structure

Figures 8-14 and 8-15 bring up one of the remaining great unsolved questions of genetics: Is a eukaryotic chromosome basically a single DNA molecule surrounded by a protein matrix? Two things strongly suggest that this is, in fact, the case. First, if there were many DNA molecules in the chromosome (whether they were side by side, end to end, or randomly oriented), it would be almost impossible for the chromosome to replicate semiconservatively (with all the label going into one chromatid, as in Taylor’s results). Studies on isolated chromosomes and long DNA molecules are consist-ent with the suggestion that each chromatid is a single molecule of DNA. The second fact supporting a single-molecule hypothesis is that DNA and genes behave as though they are attached end to end in a single string, or thread, that we call a linkage group. All genetic linkage data (Chapter 5) tell us that we need nothing more than a single linear array of genes per chromosome to explain the genetic facts. It has been convincingly demonstrated that a chromosome or a chromatid in fact contains just one DNA molecule, as we saw in Chapter 3.

Replication fork

A 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. In 1963, John Cairns tested this prediction by allowing replicating DNA in bacterial cells to incorporate tritiated thymidine. Theoretically, each newly synthesized daughter molecule should then contain one radioactive (“hot”) strand and another nonradioactive (“cold”) strand. After varying intervals and varying numbers of replication cycles in a “hot” medium, Cairns extracted the DNA from the cells, put it on a slide, and autoradiographed it for examination under the light microscope. After one replication cycle in [³H]thymidine, rings of dots appeared in the autoradiograph. Cairns interpreted these rings as shown in Figure 8-17. It is also apparent from Figure 8-17 that the bacterial chromosome is circular—a fact that also emerged from genetic analysis described earlier (Chapter 7).

Figure 8-17

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 8-18 could be made. Cairns saw all sizes of these moon-shaped, autoradiographic patterns, corresponding to the progressive movement of the replication zipper, or fork, around the ring. Structures of the sort shown in Figure 8-18 are called theta ( θ) structures.

Figure 8-18

Rolling-circle replication

The replication of some circular molecules, such as plasmids and certain viruses, proceeds by the mechanism depicted in Figure 8-19. Here, a nuclease cut provides a free 3′-OH end onto which nucleotides are added. As can be seen from Figure 8-19, as the synthesis proceeds, the other end of the template strand is displaced from the double-stranded circle and then copied. We can envision this displacement as the strand rolling off the circle. Because there is no termination point, synthesis often continues beyond a single circle unit, producing concatamers (a series of linked chains) of several circle lengths, which are then processed by recombination to yield normal-length circles.

Figure 8-19

Rolling-circle replication. Newly synthesized DNA is light blue. The displaced strand is replicated discontinuously, as described in the text. (After D. L. Hartl and E. W. Jones, Genetics: Principles and Analysis, 4th ed. Jones and Bartlett, 1998.)