Mispair-specific recruitment of Mlh1-Pms1 identifies repair substrates of Saccharomyces cerevisiae Msh2-Msh3 [Enzymology]

February 18th, 2014 by Srivatsan, A., Bowen, N., Kolodner, R. D.

DNA mismatch repair (MMR)a is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here, we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3 and +4 insertion/deletions and CC, AA and possibly GG mispairs whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent MMR in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6 and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions.