15. Controlling the direction of mismatch repair

14.15 Controlling the direction of mismatch repair

When a structural distortion is removed from DNA, the wild Vtype sequence is restored. In most cases, the distortion is due to the creation of a base that is not naturally found in DNA, and which is therefore recognized and removed by the repair system.

A problem arises if the target for repair is a mispaired partnership of (normal) bases created when one was mutated. The repair system has no intrinsic means of knowing which is the wild-type base and which is the mutant! All it sees are two improperly paired bases, either of which can provide the target for excision repair.

If the mutated base is excised, the wild-type sequence is restored. But if it happens to be the original (wild Vtype) base that is excised, the new (mutant) sequence becomes fixed. Often, however, the direction of excision repair is not random, but is biased in a way that is likely to lead to restoration of the wild Vtype sequence.

Some precautions are taken to direct repair in the right direction. For example, for cases such as the deamination of 5-methyl-cytosine to thymine, there is a special system to restore the proper sequence. The deamination generates a G PT pair, and the system that acts on such pairs has a bias to correct them to G PC pairs (rather than to A PT pairs).

The VSP system undertakes this reaction, and it includes the mutL,S system that removes T from both G PT and C PT mismatches.

Figure 14.32 Preferential removal of bases in pairs that have oxidized guanine is designed to minimize mutations.

The mutT,M,Y system handles the consequences of oxidative damage. A major type of chemical damage is caused by oxidation of G to 8-oxo-G. Figure 14.32 shows that the system operates at three levels. MutT hydrolyzes the damaged precursor (8-oxo-dGTP), which prevents it from being incorporated into DNA. When guanine is oxidized in DNA, its partner is cytosine; and MutM preferentially removes the C from 8-oxo VG:C pairs. Oxidized guanine mispairs with A, and so when 8-oxo-G survives and is replicated, it generates an 8-oxo VG:A pair. Thus MutY removes A from these pairs. MutM and MutY are glycosylases that directly remove a base from DNA. This creates a apurinic site that is recognized by an endonuclease whose action triggers the involvement of the excision repair system.

When mismatch errors occur during replication in E. coli, it is possible to distinguish the original strand of DNA. Immediately after replication of methylated DNA, only the original parental strand carries the methyl groups. In the period while the newly synthesized strand awaits the introduction of methyl groups, the two strands can be distinguished.

Figure 13.30 Replication of methylated DNA gives hemimethylated DNA, which maintains its state at GATC sites until the Dam methylase restores the fully methylated condition.

This provides the basis for a system to correct replication errors. The dam gene codes for a methylase whose target is the adenine in the sequence ^GATC_CTAG (see Figure 13.30). The hemimethylated state is used to distinguish replicated origins from nonreplicated origins. The same target sites are used by a replication-related repair system.

Figure 14.33 GATC sequences are targets for the Dam methylase after replication. During the period before this methylation occurs, the nonmethylated strand is the target for repair of mismatched bases.

Figure 14.33 shows that DNA containing mismatched base partners is repaired preferentially by excising the strand that lacks the methylation. The excision is quite extensive; mismatches can be repaired preferentially for >1 kb within a d(GATC) site. The result is that the newly synthesized strand is corrected to the sequence of the parental strand.

Figure 14.34 MutS recognizes a mismatch and translocates to a GATC site. MutH cleaves the unmethylated strand at the GATC. Endonucleases degrade the strand from the GATC to the mismatch site.

E. coli dam ^V mutants show an increased rate of spontaneous mutation. This repair system therefore helps reduce the number of mutations caused by errors in replication. It consists of several proteins, coded by the mut genes (which also participate in VSP repair) MutS binds to the mismatch and is joined by MutL. MutS can use two DNA-binding sites, as illustrated in Figure 14.34. The first specifically recognizes mismatches. The second is not specific for sequence or structure, and is used to translocate along DNA until a GATC sequence is encountered. Hydrolysis of ATP is used to drive the translocation. Because MutS is bound to both the mismatch site and to DNA as it translocates, it creates a loop in the DNA.

Recognition of the GATC sequence causes the MutH endonuclease to bind to MutSL. The endonuclease then cleaves the unmethylated strand. This strand is then excised from the GATC site to the mismatch site. The excision can occur in either the 5′ V3′ direction (using RecJ or exonuclease VII) or in the 3′ V5′ direction (using exonuclease I), assisted by the helicase UvrD. The new DNA strand is synthesized by DNA polymerase III.

The msh repair system of S. cerevisiae is homologous to the E. coli mut system. MSH2 provides a scaffold for the apparatus that recognizes mismatches. MSH3 and MSH6 provide specificity factors. The MSH2 VMSH3 complex binds mismatched loops of 2 V4 nucleotides, and the MSH2 VMSH6 complex binds to single base mismatches or insertions or deletions. Other proteins are then required for the repair process itself.