Muts Beta Abundance And Msh3 Atp Hydrolysis Activity Are Important Drivers Of Ctg Circle Cag Repeat Expansions

CTG circle CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutS beta (Msh2-Msh3 heterodimer). To evaluate properties of MutS beta that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutS beta, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutS beta. Msh3(-/-) cells are severely defective for CTG circle CAG repeat expansions but show full activity on contractions. Msh3(-/-) cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutS beta on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutS beta complex, indicating that MutS beta abundance drives expansions. An ATPasedefective Msh3 expressed at normal levels was as defective in expansions as Msh3(-/-) cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG circle CAG expansions are limited by the abundance of MutS beta and rely heavily on Msh3 ATPase function.