Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

Abstract

The major eukaryotic mismatch repair (MMR) pathway requires Msh2-Msh6, which, like E. coli MutS, binds to and participates in repair of the two most common replication errors, single base-base and single base insertion-deletion mismatches. For both types of mismatches, the side chain of E. coli Glu38 in a conserved Phe-X-Glu motif interacts with a mismatched base. The Oε of Glu38 forms a hydrogen bond with either the N7 of purines or the N3 of pyrimidines. We show here that changing E. coli Glu38 to alanine results in nearly complete loss of repair of both single base-base and single base deletion mismatches. In contrast, a yeast strain with alanine replacing homologous Glu339 in Msh6 has nearly normal repair for insertion-deletion and most base-base mismatches, but is defective in repairing base-base mismatches characteristic of oxidative stress, e.g., 8-oxo-G•A mismatches. The results suggest that bacterial MutS and yeast Msh2-Msh6 differ in how they recognize and/or process replication errors involving undamaged bases, and that Glu339 in Msh6 may have a specialized role in repairing mismatches containing oxidized bases.

Saccharomyces cerevisiae Msh2-Msh6 DNA binding kinetics reveal a mechanism of targeting sites for DNA mismatch repair

“…We also detected less stable interaction between Msh2-Msh6 E339A and G:T, while þT was unaffected. These observations are consistent with in vivo data indicating that Msh6 Glu339 is involved in repair of mismatched base pairs, but is dispensable for single base IDLs (43). In E. coli MutS, E38A mutation causes near loss of MMR (36,41,43); it has been hypothesized that MutS E38A is unable to form ATP-bound complexes with DNA (36) or its poor selectivity limits MMR at bona fide targets (42), which are both likely mechanisms based on our study of Msh2-Msh6 E339A .…”

Mechanism of MutS Searching for DNA Mismatches and Signaling Repair

“…In addition, the crystal structures of wild type and E38A E. coli MutS are very similar to one another (22). Despite this lack of differences, E. coli MutS-E38A is severely compromised for repair of base-base mismatches in vivo (21)(22)(23) and defective in the ATP-induced formation of the mobile clamp state in vitro (22). The latter finding led to the proposal that formation of a hydrogen bond between Glu and a mismatched base causes a post-recognition conformational change in the MutS mismatch complex that inhibits ATP hydrolysis and promotes formation of a mobile MutS clamp that signals repair.…”

“…Consistent with this hypothesis, mutation of the homologous residue in E. coli (E38A) or yeast (yMsh2-Msh6-E339A) to Ala results in different mutation rates for base-base mismatches and IDLs. Specifically, both E. coli MutS-E38A and yeast yMsh2-Msh6-E339A exhibit a significant increase in mutations resulting from base-base mismatches (21-23, 36), whereas E. coli MutS-E38A exhibits a lower mutation frequency for frameshift mutations than for base-base mismatches (indicating better repair of IDLs such as the T-bulge relative to mismatches) (23), and yMsh2-Msh6-E339A displays no mutator phenotype for frameshift mutations (20,23,36). The in vivo results are thus consistent with our prediction that a reduction in the population of the unbent state would lead to reduced repair and strongly support our proposal that formation of the unbent URC is essential for signaling DNA repair.…”

Functional Studies and Homology Modeling of Msh2-Msh3 Predict that Mispair Recognition Involves DNA Bending and Strand Separation

“…While the E. coli E38 residue of the F-X-E motif is absolutely required for interaction with mispaired bases or 1-nucleotide insertions, or the corresponding residue E339 in S. cerevisiae Msh6 is dispensable for the repair of insertion/deletions and most base-base mispairs (14). While a similar F-X-E pattern is present in the Msh3 homologs, S. cerevisiae Msh3 E164 is not predicted to interact with the mispaired base or ϩ1 frameshift mispair in the homology model and the E164A mutant appears to be wild type in the frameshift and loop repair assays.…”