Abstract
DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.
Highlights
DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors
Five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR
When WT Msh2–Msh6 is bound to a mispair, ATP binding at both the high- (Msh6) and low (Msh2)-affinity nucleotidebinding sites triggers a conformational change from a relatively static mispair recognition complex to a clamp that can slide along the DNA [21,22,23,24, 32, 55]
Summary
When WT Msh2–Msh is bound to a mispair, ATP binding at both the high- (Msh6) and low (Msh2)-affinity nucleotidebinding sites triggers a conformational change from a relatively static mispair recognition complex to a clamp that can slide along the DNA [21,22,23,24, 32, 55]. A genetic screen for dominant mutations in MSH6 recovered several msh mutations that affected residues clustered around the Msh ATPase site [51] These mutations cause large increases in mutation rates, including increased frameshift reversion rates when present as single mutations at the MSH6 locus and when present on a low-copy ARS CEN plasmid in an otherwise WT strain [21, 30, 51, 52]. Previous analysis of the Walker A Gly msh mutations showed that these mutations did not cause an increased mutation rate in a WT strain when present on an ARS CEN plasmid [46, 47], which is consistent with our results, but did cause a very small increase in mutation rate when present in a heterozygous diploid S. cerevisiae strain These mutations and a Walker A Gly msh mutation were dominant when overexpressed using a GAL10 promoter on a high-copy-number 2-m plasmid [28, 46, 47]. At concentrations of ATP (2.5 mM) that exceed the Kd for ATP binding by both the Msh and Msh nucleotide-binding sites, the Walker A Lys mutant Msh2–Msh complexes supported 40 –75% of the level of
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