A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine
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As stated previously, bacteria such as E. coli are capable of dividing once every 30 minutes, making it relatively easy to screen large populations to find a rare mutant cell that is altered in a specific process. One interesting class of mutants contains alterations in so-called mutator genes, which greatly increase the rate of spontaneous mutation when they are inactivated. Not surprisingly, one such mutant makes a defective form of the 3′-to-5′ proofreading exonuclease that is a part of the DNA polymerase enzyme (see Figures 5-9 and 5-10). When this activity is defective, the DNA polymerase no longer proofreads effectively, and many replication errors that would otherwise have been removed accumulate in the DNA.
The study of other E. coli mutants exhibiting abnormally high mutation rates has uncovered another 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 that results from the misfit between noncomplementary base pairs. But if the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakingly “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 mismatch, excision of the segment of DNA containing the mismatch from the newly synthesized strand, and resynthesis of the excised segment using the old strand as a template—thereby removing the mismatch. This strand-directed mismatch repair system reduces the number of errors made during DNA replication by an additional factor of 102 (see Table 5-1, p. 243).
A similar mismatch proofreading system functions in human cells. The importance of this system is indicated by the fact that individuals who inherit one defective copy of a mismatch repair gene (along with a functional gene on the other copy of the chromosome) have a marked predisposition for certain types of cancers. In a type of colon cancer called hereditary nonpolyposis colon cancer (HNPCC), spontaneous mutation of the remaining functional gene produces a clone of somatic cells that, because they are deficient in mismatch proofreading, accumulate mutations unusually rapidly. Most cancers arise from cells that have accumulated multiple mutations (discussed in Chapter 23), 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 that both copies would mutate in the same cell.
In eucaryotes, the mechanism for distinguishing the newly synthesized strand from the parental template strand at the site of a mismatch does not depend on DNA methylation. Indeed, some eucaryotes—including yeasts and Drosophila—do not methylate any of their DNA. Newly synthesized DNA strands are known to be preferentially nicked, and biochemical experiments reveal that such nicks (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand in a eucaryotic cell (Figure 5-23).
Figure 5-23
A model for strand-directed mismatch repair in eucaryotes. (A) The two proteins shown are present in both bacteria and eucaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once a nick is found, (more...)