DNA Replication Areas In Chromosomes
After considering the enzymology of the DNA replication and repair processes, we turn to more biological questions. As a first step, it is useful to learn the number of DNA synthesis regions per bacterial or eukaryotic chromosome. To see why this is important, consider the two extremes. On one hand, a chromosome could be duplicated by a single replication fork traversing the entire stretch of a DNA molecule. On the other hand, many replication points per chromosome could function simultaneously. The requisite elongation rates and regulation mecha-nisms would be vastly different in the two extremes. Furthermore, if many replication points functioned simultaneously, they could be either scattered over the chromosome or concentrated into localized replica-tion regions.
The most straightforward method for determining the number of replication regions on a chromosome is electron microscopy. This is possible for smaller bacteriophage or viruses, but the total amount of DNA contained in a bacterial chromosome is far too great to allow detection of the few replication regions that might exist. The situation is even worse for chromosomes from eukaryotes because they contain as much as one hundred times the amount of DNA per chromosome as bacteria. The solution to this problem is not to look at all the DNA but to look at just the DNA that has been replicated in the previous minute. This can easily be done by autoradiography. Cells are administered highly radioactive thymidine, and a minute later the DNA is extracted and gently spread on photographic film to expose a trail which, upon development, displays the stretches of DNA that were synthesized in the presence of the radioactivity.
The results of such autoradiographic experiments show that cultured mammalian cells contain DNA synthesis origins about every 40,000 to 200,000 base pairs along the DNA. In bacteria, the result was different;
Figure 3.11 Sketch of an electronmicrograph of an autoradiograph of a partially replicated bacterial chromo-some that has been labeled with radioactive precursor for 40 minutes before extraction of DNA and prepa-ration.
administration of a short pulse of radioactive thymidine was unneces-sary. When thymidine was provided for more than one doubling time, the entire chromosome of the bacterium could be visualized via the exposed photographic grains. Two startling facts were seen: the chro-mosome was circular, and it possessed only one or two replication regions (Fig. 3.11).
The existence of a circular DNA molecule containing an additional segment of the circle connecting two points, the theta form, was inter-preted as showing that the chromosome was replicated from an origin by one replication region that proceeded around the circular chromo-some. It could also have been interpreted as demonstrating the existence of two replication regions that proceeded outward in both directions from a replication origin. Some of the original autoradiographs pub-lished by Cairns contain suggestions that the DNA is replicated in both directions from an origin. This clue that replication is bidirectional was overlooked until the genetic data of Masters and Broda provided solid and convincing data for two replication regions in the E. coli chromo-some.
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