Abstract
MutSalpha (MSH2/MSH6) and MutSbeta (MSH2/MSH3) are eukaryotic mismatch recognition proteins that preferentially process base-base and small insertion/deletion (ID) mispairs, respectively, despite the fact that cells contain a MutSalpha:MutSbeta ratio of 10:1. To explore the mechanism underlying the differential mismatch recognition by these two proteins, purified human MutSalpha and MutSbeta were analyzed individually and competitively for their abilities to interact with a T-G and an ID substrate. We show that MutSalpha has K(D) values of 26.5 and 38.2 nm for the G-T and ID substrates, respectively, and that MutSbeta has K(D) values of 76.5 and 23.5 nm for G-T and ID, respectively. Consistent with these results, competitive binding assays revealed the following relative binding affinities: MutSbeta-ID > MutSalpha-T-G > MutSalpha-ID >> MutSbeta-T-G. Interestingly, binding of MutSbeta to ID heteroduplexes is greatly stimulated when the MutSalpha:MutSbeta ratio is > or = 10. Distinct ATP/ADP binding and ATPase activities of MutSalpha and MutSbeta were also observed. In the absence of DNA, ADP binding and ATPase activities of MutSbeta are significantly higher than those of MutSalpha. However, interaction with DNA significantly stimulates the MutSalpha ATPase activity and reduces the MutSbeta ATPase activity, the consequence being that both proteins exhibit the same level of hydrolytic activity. We conclude that the preferential processing of base-base and ID heteroduplexes by MutSalpha and MutSbeta is determined by their significant differences in ATPase activity, ADP binding activity, and high cellular MutSalpha:MutSbeta ratio.
Highlights
Manner dependent on MutL family proteins and several other protein factors, and DNA repair synthesis by replicative DNA polymerases in concert with factors involved in DNA replication [1]
At least two mismatch recognition proteins, MutS␣ and MutS, have been identified in eukaryotic cells, and each of them is a heterodimeric complex. Both genetic and biochemical studies suggest that MutS␣ and MutS have partially overlapping functions, with MutS␣ targeting base-base mismatches and 1–2-nucleotide ID mispairs and MutS targeting Ն2-nt but Յ16-nt ID heteroduplexes [5,6,7,8]
In addition to the mismatch binding activity, all MutS proteins, from E. coli to humans, contain an ATPase activity and ATP and ADP binding activities [12,13,14]. Both the nucleotide binding and ATPase activities of the MutS family proteins are essential for MMR [15,16,17,18,19], but how these activities work in MMR is not fully understood
Summary
Manner dependent on MutL family proteins and several other protein factors, and DNA repair synthesis by replicative DNA polymerases in concert with factors involved in DNA replication [1]. ATPase activity of MutS␣ and MutS was assayed in 20-l reactions containing 50 mM Tris-HCl (pH 8.0), 110 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, [␥-32P]ATP, and the indicated amount of proteins and DNA substrates.
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