The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

DNA mismatch repair (MMR) acts on mispairs arising from DNA-replication errors, formation of homologous recombination intermediates, and some chemically modified DNA bases (1–3). During MMR, mispair recognition by MutS homologs, primarily Msh2-Msh6 and Msh2-Msh3 in eukaryotes (4–8), is required to recruit MutL homologs to mispaired DNA, primarily Mlh1-Pms1 in eukaryotes (called MLH1-PMS2 in humans) (1–3, 9). In organisms other than Escherichia coli and related bacteria (10), the MutL homologs have an endonuclease activity that specifically nicks double-stranded DNA on strands containing pre-existing nicks (11–13). Nicking by Mlh1-Pms1 in vitro is required for Exo1-mediated repair on substrates with a nick 3′ to the mispair, as formation of a strand-specific nick 5′ to the mispair allows the 5′–3′ exonuclease activity of Exo1 to excise the mispair (11–14). The absolute requirement of this Mlh1-Pms1 nicking activity in vivo is not well understood, as both 5′ and 3′ nicks relative to mispairs are likely already present on newly synthesized DNA strands (15, 16). One proposal suggests that Mlh1-Pms1 activity maintains single-stranded discontinuities, which appear to identify the newly synthesized strand, even in the presence of the competing activities, like DNA ligation and gap filling by DNA polymerases (15, 17).MutL homologs are comprised of an N-terminal GHKL family adenosine triphosphatase (ATPase) domain, a carboxyl-terminal dimerization domain, and a predicted unstructured linker domain that connects the folded N- and carboxyl-terminal domains (18–21). In Saccharomyces cerevisiae, the unstructured linkers of Mlh1 and Pms1 are ∼150 and 250 amino acids long, respectively (22). These linkers have a biased sequence composition with reduced hydrophobic amino acids, like the large (>50 amino acid) intrinsically disordered regions (IDRs) present in many proteins (23–25). IDRs often mediate intermolecular interactions, play functional roles, and sometimes become ordered when bound to partners (23–25).MutL homologs, including Mlh1-Pms1, form DNA-bound rings called sliding clamps following loading by MutS homologs, ATP binding, and dimerization of the N-terminal ATPase domains; these rings rapidly diffuse along the DNA axis (26–30). The extended length of the unstructured interdomain linkers has been suggested to allow these MutL homolog clamps to migrate past protein–DNA complexes, which are normally a barrier to MutS homolog clamps, although Msh2-Msh3 clamps appear to be able to open and close on encountering a protein–DNA complex and hop over it (26–29, 31, 32). Remarkably, cleavage of the S. cerevisiae Mlh1 linker in vivo causes increased mutation rates, suggesting that intact sliding clamps are important for MMR (22). The importance of the combined lengths of the Mlh1 and Pms1 linkers in vivo is suggested by the synergistic increases in mutation rate that have been observed when combining S. cerevisiae mlh1 and pms1 mutations that shorten the linkers (26). In contrast, some linker missense mutations, which do not alter linker lengths, cause MMR defects (22, 33–35). Moreover, deletions within the S. cerevisiae Mlh1 linker tend to cause MMR defects, whereas deletions in the S. cerevisiae Pms1 linker tend not to, except for the pms1-Δ390–610 deletion that eliminates almost the entire Pms1 linker, resulting in a mutant complex that cannot be recruited by Msh2–Msh6 to mispair-containing DNA and fails to bind to DNA under low ionic strength conditions (22). Together, the data suggest that length is only one requirement for the Mlh1 and Pms1 linkers and that the Mlh1 and Pms1 linkers differ in importance for MMR.Here, we have identified a motif in the Mlh1 linker, which spans residues 391–415, that is conserved from S. cerevisiae to humans and is required for MMR. Mutation of two of the residues in this motif, R401 and I409, to alanine caused an MMR defect, as did short deletions affecting other partially conserved residues within the motif. We found that the motif was functional when moved to different positions on the Mlh1 linker and when the distances between motif and the folded N- and carboxyl-terminal domains were altered. Moreover, moving a copy of the motif to the Pms1 subunit complemented the MMR defect caused by loss of the motif in Mlh1; in addition, swapping the Mlh1 linker with the Pms1 linker supported MMR. Mutant Mlh1-Pms1 complexes with amino acid substitutions in the conserved Mlh1 motif could not support reconstituted MMR reactions in vitro and were defective for Mlh1-Pms1 endonuclease activity but were recruited to mispair-containing DNA by Msh2-Msh6. Peptides encoding the conserved motif, but not control peptides, inhibited wild-type Mlh1-Pms1 endonuclease activity. Consistent with these observations, increased levels of Pms1-4GFP foci, which are MMR intermediates (36), were caused by mutations disrupting the conserved Mlh1 motif, similar to other mutations that reduce Mlh1-Pms1 endonuclease activity (36–38). Mutations of the motif were observed in human cancers, and these mutations disrupted MMR in vivo when modeled in the S. cerevisiae MLH1 gene. Taken together, these data are consistent with a requirement of the Mlh1 linker motif for Mlh1-Pms1 endonuclease activity in MMR, which could be due to an interaction of the motif with the DNA substrate, with the endonuclease active site, and/or with the endonuclease-activating PCNA.