One fundamental approach in the study of complex systems is to determine the minimal set of purified components that will carry out the process under investigation. In the case of DNA synthesis, the relatively loose association of the proteins involved created problems. How can one of the components be assayed so that its purification can be monitored if all the components must be present for DNA synthesis to occur? We will see that the problem was solved, but the purification of the many proteins required for DNA synthesis was a monumental task that occupied biochemists and geneticists for many years. By contrast, the machinery of protein synthesis was much easier to study because most of it is bound together in a ribosome.
A basic problem facing an organism is maintaining the integrity of its DNA. Unlike protein synthesis, in which one mistake results in one altered protein molecule, or RNA synthesis, in which one mistake ultimately shows up just in the translation products of a single messen-ger RNA, an uncorrected mistake in the replication of DNA can last forever. It affects every descendant every time the altered gene is expressed. Thus it makes sense for the mechanism of DNA synthesis to have evolved to be highly precise. There is only one real way to be precise, and that is to check for and correct any errors a number of times. In the replication of DNA, error checking of an incorporated nucleotide could occur before the next nucleotide is incorporated, or checking for errors could occur later. Apparently, checking and correct-ing occurs at both times. In the case of bacteria, and at least in some eukaryotes, the replication machinery itself checks for errors in the process of nucleotide incorporation, and an entirely separate machinery detects and corrects errors in DNA that has already been replicated. Retroviruses like HIV are an interesting exception. These have small genomes and they need a high spontaneous mutation rate in order to evade their host’s immunological surveillance system.
Generally, DNA must also maintain its structure against environ-mental assaults. Damage to the bases of either DNA strand could lead to incorrect base-pairing upon the next round of DNA replication. A number of enzymes exist for recognizing, removing, and replacing damaged bases.
Since many cell types can grow at a variety of rates, sophisticated mechanisms have developed to govern the initiation of DNA replication. In both bacteria and eukaryotic cells, it is the initiation of replication that is regulated, not the elongation. Although such a regulation system seems difficult to coordinate with cell division, the alternative mecha-nisms for regulating the rate of DNA synthesis are more complicated. In principle, the DNA elongation rate could be adjusted by changing the concentrations of many different substrates within the cell. This, how-ever, would be most difficult because of the interconnected pathways of nucleotide biosynthesis. Alternatively, the elongation rate of the DNA polymerase itself could be variable. This too, would be most difficult to manage and still maintain high fidelity of replication. Another problem closely tied to DNA replication is the segregation of completed chromo-somes into daughter cells. Not surprisingly, this process requires a complicated and specialized machinery.
After examining the basic problems generated by the structure of DNA, we discuss the enzymology of DNA synthesis. Then we mention the meth-ods cells use to maximize the stability of information stored in DNA. The second half of the chapter concerns physiological aspects of DNA synthesis. Measurement of the number of functioning replication areas per chromosome, the speed of DNA replication, and the coupling of cell division to DNA replication are covered.