Differential Biochemical Activities of MutSα and MutSβ in mismatch Recognition and Nucleotide Binding and Hydrolysis

MutSα (MSH2‐MSH6) and MutS ? (MSH2‐MSH3) are eukaryotic mismatch recognition proteins that are preferentially responsible for processing base‐base and small insertion/deletion (ID) mismatches in vivo, respectively, despite the fact that cells express 10‐fold more MutSα than MutS ?. To understand the mechanism underlying the differential mismatch recognitions by these two proteins, purified human MutSα and MutS ? were analyzed individually and competitively for their abilities to interact with a T‐G and an ID substrates. We show that MutSα has a K D of 26.5 nM and 38.2 nM for the G‐T and ID substrates, respectively, and MutS ? has a K D of 76.5 nM and 23.5 nM for G‐T and ID, respectively. Consistent with these results, competitive binding assays revealed the following relative binding affinities: MutS ?‐ID > MutSα‐T‐G > MutSα‐ID > MutS ?‐T‐G. Interestingly, MutS ?‐ID interaction can be greatly stimulated when the MutSα:MutS ? ratio is = 10, justifying why cells maintain a high MutSα:MutS ? ratio. Distinct ATP binding and hydrolysis activities of MutSα and MutS ? were observed. For MutSα, the MSH6 subunit preferentially binds ATP, but the MSH2 subunit preferentially binds ADP. In the case of MutS ?, both the MSH3 and MSH2 subunits interact with ATP and ADP, despite the fact that MSH3 has a higher affinity for ATP than MSH2 and that MSH2 possesses a higher affinity for ADP than MSH3. We also find that MutSα ATPase activity can be stimulated by DNA substrates, but DNA inhibits MutS ? ATPase activity. These distinct biochemical activities of MutSα and MutSβ may contribute to their preferential recognition of base‐base mismatches and ID mispairs, respectively.