Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA

The interaction of the Saccharomyces cerevisiae MSH2-MSH6 complex with misp aired bases was analyzed using gel mobility shift assays and surface plasmo n resonance methods. Under equilibrium binding conditions, MSH2-MSH6 bound to homoduplex DNA with a K-d of 3.9 nM and bound oligonucleotide duplexes c ontaining T:G, +1, +2, +4, and +10 insertion/deletion loop (IDL) mispairs w ith K-d values of 0.20, 0,25, 11, 3.2, and 0.55 nM, respectively. Competiti on binding experiments using 65 different substrates repealed a 10-fold ran ge in mispair discrimination. In general, base-base mispairs and a +1 inser tion/deletion mispair were recognized better than intermediate sized insert ion/deletion mispairs of 2-8 bases. Larger IDL mispairs (>8 bases) were rec ognized almost as well as the +1 IDL mispair. Recognition of mispairs by MS H2-MSH6 was influenced by sequence context, with the 6-nucleotide region su rrounding the mispair being primarily responsible for influencing mispair r ecognition. Effects of sequences as far away as 15 nucleotides were also ob served, Differential effects of ATP on the stability of MSH2-MSH6-mispair c omplexes suggested that base-base mispairs and the smaller IDL mispairs wer e recognized by a different binding mode than larger IDL mispairs, consiste nt with genetic experiments indicating that MSH2-MSH6 functions primarily i n the repair of base-base and small IDL mispairs.