The study of other E. coli mutants exhibiting abnormally high mutation rates has uncovered a proofreading system that removes replication errors made by the polymerase that have been missed by the proofreading exonuclease. This strand-directed mismatch repair system detects the potential for distortion in the DNA helix from the misfit between noncomplementary base pairs.
If the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakenly “correct” the original template strand to match the error exactly half the time, thereby failing to lower the overall error rate. To be effective, such a proofreading system must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred.
The strand-distinction mechanism used by the mismatch proofreading system in E. coli depends on the methylation of selected A residues in the DNA. Methyl groups are added to all A residues in the sequence GATC, but not until some time after the A has been incorporated into a newly synthesized DNA chain. As a result, the only GATC sequences that have not yet been methylated are in the new strands just behind a replication fork. The recognition of these unmethylated GATCs allows the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed. The three-step process involves recognition of a newly synthesized strand, excision of the portion containing the mismatch, and resynthesis of the excised segment using the old strand as a template. This strand-directed mismatch repair system reduces the number of errors made during DNA replication by an additional factor of 100 to 1000 (see Table 5–1, p. 244).
A similar mismatch proofreading system functions in eukaryotic cells but uses a different strategy to distinguish the new strand from the old (Figure 5–19). Newly synthesized lagging-strand DNA transiently contains nicks (before they are sealed by DNA ligase) and such nicks (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand. This strategy also requires that the newly synthesized DNA on the leading strand be transiently nicked; how this occurs is uncertain.
The importance of mismatch proofreading in humans is seen in individuals who inherit one defective copy of a mismatch repair gene (along with a functional gene on the other copy of the chromosome). These people have a marked predisposition for certain types of cancers. For example, in a type of colon cancer called hereditary nonpolyposis colon cancer (HNPCC), spontaneous mutation of the one functional gene produces a clone of somatic cells that, because they are deficient in mismatch proofreading, accumulate mutations unusually rapidly. Most cancers arise in cells that have accumulated multiple mutations (see pp. 1096–1097), and cells deficient in mismatch proofreading therefore have a greatly enhanced chance of becoming cancerous. Fortunately, most of us inherit two good copies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely for both copies to become mutated in the same cell.
As a replication fork moves along double-strand DNA, it creates what has been called the “winding problem.” The two parental strands, which are wound around each other, must be unwound and separated for replication to occur. For every 10 nucleotide pairs replicated at the fork, one complete turn of the parental double helix must be unwound. In principle, this unwinding can be achieved by rapidly rotating the entire chromosome ahead of a moving fork; however, this is energetically highly unfavorable (particularly for long chromosomes) and, instead, the DNA in front of a replication fork becomes overwound (Figure 5–20). The over-winding, in turn, is continually relieved by proteins known as DNA topoisomerases.
A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.