A special situation occurs in vertebrate DNA, in which selected C nucleotides are methylated at specific CG sequences that are associated with inactive genes (discussed in Chapter 7). The accidental deamination of these methylated C nucleotides produces the natural nucleotide T (Figure 5–43B) in a mismatched base pair with a G on the opposite DNA strand. To help in repairing deaminated methylated C nucleotides, a special DNA glycosylase recognizes a mismatched base pair involving T in the sequence T-G and removes the T. This DNA repair mechanism must be relatively ineffective, however, because methylated C nucleotides are exceptionally common sites for mutations in vertebrate DNA. It is striking that, even though only about 3% of the C nucleotides in human DNA are methylated, mutations in these methylated nucleotides account for about one-third of the single-base mutations that have been observed in inherited human diseases.
If a cell’s DNA suffers heavy damage, the repair mechanisms that we have discussed are often insufficient to cope with it. In these cases, a different strategy is called into play, one that entails some risk to the cell. The highly accurate replicative DNA polymerases stall when they encounter damaged DNA, and in emergencies cells employ versatile, but less accurate, backup polymerases, known as translesion polymerases, to replicate through the DNA damage.
Human cells have seven translesion polymerases, some of which can recognize a specific type of DNA damage and correctly add the nucleotide required to restore the initial sequence. Others make only “good guesses,” especially when the template base has been extensively damaged. These enzymes are not as accurate as the normal replicative polymerases when they copy a normal DNA sequence. For one thing, the translesion polymerases lack exonucleolytic proofreading activity; in addition, many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially. Presumably for this reason, each such translesion polymerase is given a chance to add only one or a few nucleotides before the highly accurate replicative polymerase resumes DNA synthesis.
Despite their usefulness in allowing heavily damaged DNA to be replicated, these translesion polymerases do, as noted above, pose risks to the cell. They are probably responsible for most of the base-substitution and single-nucleotide deletion mutations that accumulate in genomes; although they generally produce mutations when copying damaged DNA (see Figure 5–40), they probably also create mutations—at a low level—on undamaged DNA. Clearly, it is important for the cell to tightly regulate these polymerases, releasing them only at sites of DNA damage. Exactly how this happens for each translesion polymerase remains to be discovered, but a conceptual model is given in Figure 5–44. The principle of this model applies to many of the DNA repair processes discussed in this chapter: because the enzymes that carry out these reactions are potentially dangerous to the genome, they must be brought into play only at sites of damage.
An especially dangerous type of DNA damage occurs when both strands of the double helix are broken, leaving no intact template strand to enable accurate
Figure 5–44 Translesion DNA polymerases can use damaged templates. According to this model, a replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated—see Figure 3–69) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play.