In vitro studies of MutSalpha protein in mismatch excision repair and DNA damage signaling

DNA mismatch repair (MMR) system plays a key role to detect and correct DNA mismatches generated during replication and recombination. Mutations in MMR genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC). In eukaryotes, the first step of MMR is the recognition of mismatches by MutSalpha, which is a heterodimer of MSH2 and MSH6. The loss of MSH2 or MSH6 results in the accumulation of somatic mutations in the genomes of tumor cells and resistance to the genotoxic effects of a variety of DNA damaging agents. Recently, several separation‐of‐function mutations have been identified in mice in which MMR is impaired but the apoptotic response to DNA damaging agents is retained. The corresponding human mutations, MSH2 G674A and MSH6 T1219D have been identified as being HNPCC alleles. We are characterizing the molecular defects in these two mutant proteins. Both MSH2G674A‐MSH6wt and MSH2wt‐MSH6T1219D proteins fail to carry out MMR in an in vitro assay. Also, these two mutants do not carry out “normal” excision, giving rise to shortened excision tracts that fail to remove the incorrect base. Both mutated MutSalpha proteins possess similar DNA binding activity to a G:T mismatch as wildtype MutSalpha, but are impaired in the removal of mismatched bases in the presence of ATP. Furthermore, these two mutants failed to interact in SPR assays with MutLalpha protein, which is critical for multiple steps of MMR.