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DNA mismatch repair (MMR) is an evolutionary conserved process that functions to maintain genomic integrity through the correction of mismatches that have escaped proofreading. Mutations in the MMR gene Mlh1 are associated with approximately 50% of all cases of Lynch syndrome, a hereditary predisposition to colorectal cancer, through varying and largely unidentified mechanisms. Microsatellite instability (MSI) is a hallmark of such MMR deficient cancers, the identification of which is essential, as they may respond differently to chemotherapeutic drugs. The recent identification of the MLH1 variant D132H, which gives rise to Lynch syndrome cancers in the absence of MSI, makes this mutant an interesting target for analysis. In the present study, we developed a new screening assay - based on previous work with MLH1 deficient mouse embryonic fibroblasts - that allows for quick and efficient assessment of a variety of potential pathogenic mechanisms. The results from these assays, together with in vitro repair experiments, demonstrate that D132H is able to stabilize PMS2, repair base/base and insertion deletion loop mismatches, and promote the cytotoxicity of the DNA damaging agent 6-thioguanine, all of which suggest the D132H mutation does dramatically affect MMR activity. Our assay was also used to evaluate the effects of another pathogenic mutant, K616Δ, and suggest, consistent with some previous studies, that the mutation affects MLH1 expression in vivo. |
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