Multiple Factors Insulate Msh2–Msh6 Mismatch Repair Activity from Defects in Msh2 Domain I

Abstract

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2–Msh6 binds mispairs and small insertion/deletion loops, and Msh2–Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2–Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2–Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2–Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1–Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.