The mechanics of DNA mismatch repair sliding clamp progression in the prediction of clinically relevant HsMLH1 missense mutations

Abstract

Lynch Syndrome (LS), the most common inherited colorectal cancer predisposition, is primarily caused by mutations in the DNA mismatch repair (MMR) genes: HsMSH2, HsMLH1, HsMSH6 and HsPMS2. MMR proteins maintain genomic stability by repairing nucleotide mispairing errors during DNA replication. They are highly conserved members of the MutS (MSH) and MutL (MLH/PMS) proteins that play central roles in directing accurate MMR excision. MSH proteins recognize mismatched nucleotides which triggers ATP binding and formation of a sliding clamp that randomly diffuses along the DNA. Previous biophysical studies in E.coli, showed that EcMutS and EcMutL formed cascading sliding clamps on the mismatched DNA, and defined the intermediary activity of EcMutL, as a signal transmitter between the mismatch recognition and the downstream excision processes. However, the detailed mechanics of the human MLH/PMS proteins has remained a puzzle. Using real-time-single molecule imaging we show that human heterodimeric MMR proteins HsMSH2-HsMSH6 and HsMLH1-HsPMS2 exhibit identical mechanism to their bacterial homologs. HsMSH2-HsMSH6 is required to load the major human MLH/PMS heterodimer onto a DNA containing a single mismatch. In its absence, no visible HsMLH1-HsPMS2 sliding clamps were observed. MLH/PMS proteins contain a disordered region, variable in length, that connects the N-terminal ATP binding domain that interacts with HsMSH2-HsMSH6 with the C-terminal dimerization domain of the protein. Our results indicate that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to wrap around the DNA and form a stable sliding clamp, diffusing rapidly along the DNA. Furthermore, it has been shown that increasing the number of amino acids deleted in the linker domain in EcMutL and S. cerevisae ScMlh1 or ScPms1, initially block the ability to transit roadblocks on the DNA to ultimately, total MMR inhibition. MLH/PMS sliding clamps resemble a flexible donut that encircles the DNA with a relatively large donut hole. We hypothesize that any sequence of amino acids could, in principle, occupy this domain with the condition that it remained malleable and capable of wrapping around the DNA. Therefore, few if any LS pathogenic missense protein variants (MPVs) are expected within the HsMLH1 linker domain. To test this, unique missense protein variants (MPVs) located in the coding sequence of HsMLH1 were analyzed, A clear pattern was found, where non-pathogenic (26) and uncertain (366) variants were distributed ubiquitously. However, the disordered linker region did not contain any pathogenic (71) variants. We conclude that uncertain MPVs located in the linker are unlikely to be pathogenic since it is doubtful that any single amino acid substitution could influence the natural disorder that is essential for linker-domain function. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of pathogenic HsMLH1 missense mutations.