Strand discrimination in DNA mismatch repair

Abstract

DNA mismatch repair (MMR) corrects non-Watson-Crick basepairs generated by replication errors, recombination intermediates, and some forms of chemical damage to DNA. In MutS and MutL homolog-dependent MMR, damaged bases do not identify the error-containing daughter strand that must be excised and resynthesized. In organisms like Escherichia coli that use methyl-directed MMR, transient undermethylation identifies the daughter strand. For other organisms, growing in vitro and in vivo evidence suggest that strand discrimination is mediated by DNA replication-associated daughter strand nicks that direct asymmetric loading of the replicative clamp (the β-clamp in bacteria and the proliferating cell nuclear antigen, PCNA, in eukaryotes). Structural modeling suggests that replicative clamps mediate strand specificity either through the ability of MutL homologs to recognize the fixed orientation of the daughter strand relative to one face of the replicative clamps or through parental strand-specific diffusion of replicative clamps on DNA, which places the daughter strand in the MutL homolog endonuclease active site. Finally, identification of bacteria that appear to lack strand discrimination mediated by a replicative clamp and a pre-existing nick suggest that other strand discrimination mechanisms exist or that these organisms perform MMR by generating a double-stranded DNA break intermediate, which may be analogous to NucS-mediated MMR.

Keywords: DNA mismatch repair; DNA mispair; Strand discrimination.

Figure 1.. MMR must target the daughter DNA strand.

Hypothetical processing of a T:G mispair arising due to a DNA replication error can lead to multiple genetic outcomes. In MMR, excision and resynthesis target the daughter strand (gray), which eliminates the mispair prior to DNA replication and no mutant progeny are produced. If the mispair is unrepaired, then DNA replication will generate one wild-type progeny and one mutant progeny. If the parental strand (black) were to be targeted by excision and resynthesis, then the mispair would be converted into a mutation and propagated in all subsequent rounds of DNA replication.

Figure 2.. Methyl-directed MMR.

Replication of d(GATC) sites that are methylated on both strands (solid circles) generates hemi-methylated sites that distinguish the parental and daughter strands. Mispairs generated during DNA replication are recognized by the MutS homodimer that then recruits the MutL homodimer. MutL complexes (or MutS-MutL complexes) can then migrate along DNA to activate the MutH single-stranded endonuclease at hemi-methylated d(GATC) either 5’ or 3’ to the mispair. These nicks become entry sites for the recruitment and activation of the UvrD helicase that either generates a single-stranded daughter strand flap (single MutH incision) or a gap in which the mispair-proximal region of the daughter strand is removed (multiple MutH incision). Gap filling by DNA polymerase and DNA ligase completes the repair of the daughter strand.

Figure 3.. In vitro reconstituted eukaryotic MMR reactions.

A. EXO1-dependent repair of a mispair-containing substrate with a 5’ nick can proceed by mispair recognition by MSH2-MSH6 and EXO1 recruitment. Nicked strand degradation uses the 5’-3’ exonuclease activity of EXO1. B. EXO1-dependent repair of a substrate with a 3’ nick requires the MLH1-PMS2 endonuclease and its PCNA activator to generate a 5’ nick. Repair then occurs for the 5’ nick containing substrate. C. One possible repair reaction of a 3’ nicked substrate in the absence of EXO1 repair can proceed by generation of a 5’ nick by MLH1-PMS2 followed by strand displacement synthesis by DNA polymerase δ. D. Another possible repair reaction in the absence of EXO1 can proceed by the generation of multiple nicks by MLH1-PMS2 that can give rise to a gap through daughter fragment dissociation, which can then be repaired by gap filling using a DNA polymerase.

Figure 4.. Features of the C-terminal domains of MutL homologs.

A. Ribbon diagram of E. coli MutL homolog comprised of N-terminal ATPase domains (top; PDB ID 1b62 (98)) and a C-terminal dimerization domain made up of a dimerization/nuclease subdomain and a regulatory subdomain (bottom; PDB ID 1x9z (99)). B. Comparison of the C-terminal domain structures of E. coli MutL (a methyl-directed MutL without an endonuclease active site; PDB ID 1x9z (99)), B. subtilis MutL (a methyl-independent MutL with an endonuclease active site; PDB ID 3kdk (64)), Aquifex aeolicus MutL (containing an endonuclease active site but not a regulatory subdomain; PDB ID 5b42 (151)), and S. cerevisiae Mlh1-Pms1 (containing an endonuclease active site and a regulatory subdomain in the Pms1 protein; PDB ID 4e4w (154)). C. Diagram of MutLs from methyl-dependent and methyl-independent subfamilies I-III. Endonuclease domains are missing from the methyl-dependent MutL proteins, but present in the methyl-independent MutLs. The methyl-independent subfamilies II and III lack the regulatory domain involved in interaction with the β-clamp through mutation (subfamily II) or deletion (subfamily III).

Figure 5.. The MutL-directed strand specificity model for strand-specific cleavage.

A. The bacteria β-clamp (green) is loaded by the clamp loader (subunits black to grey) so that the “front” face is oriented towards the 3’ end of the discontinuous (daughter) strand. The model was generated using the E. coli β-clamp DNA complex (PDB ID 3bep (111)), the E. coli clamp loader bound to a primer-template DNA (grey; PDB ID 3glf (102)) and the β-clamp complex with clamp loader δ subunit (not shown; PDB ID 1jqj (103)). B. The orientation of the faces of the replicative clamps relative to the two strands are preserved during potentially non-strand-specific diffusion along DNA. C. To mediate daughter strand specific cleavage, clamp-bound MutL homologs (blue) must specifically cleave the strand emerging in a 5’ to 3’ direction from the “front” face of the replicative clamp (red arrows). Image generated with the B. subtilis β-clamp fused to the MutL regulatory subdomain (PDB ID 6e8d (100)).

Figure 6.. The strand-specific clamp diffusion model for strand-specific cleavage.

A. Structure of the human PCNA trimer (green surface and cartoon) bound to a double-stranded DNA fragment with the tracked strand in orange (PDB ID 6gis (106)). B. Detail of the interactions of the phosphates of the tracked DNA strand with positively charged residues primarily in one PCNA subunit. C. The bacteria β-clamp (green) is loaded onto the continuous (parental; orange) strand of a DNA duplex by the clamp loader (subunits black to grey) as shown in Figure 5A, orienting the clamps to track along the parental strand by strand-specific diffusion. D. The β-clamp (green) interaction with the regulatory subdomain of the MutL C-terminus (light blue) places the active site of the molecule (dark blue) in an appropriate position to cleave the non-tracked, daughter DNA strand (red). This model, together with strand-specific loading shown in panel C, explains how nicks, the replicative sliding clamp, and MutL homologs can mediate strand specificity. The model was generated using the structure of the B. subtilis β-clamp fused to the MutL regulatory subdomain (PDB ID 6e8d (100)), the B. subtilis MutL C-terminal domain (PDB ID 3kdk (64)), and the E. coli β-clamp DNA complex (PDB ID 3bep (111)).