DNA mismatch repair (MMR) is a highly conserved cellular process that functions in the maintenance of genomic integrity. Mutations in DNA MMR genes increase cellular mutation rates and underlie the cancer predisposition syndrome, hereditary non-polyposis colorectal cancer, also known as Lynch syndrome. The current availability of genetic testing has promoted identification of novel cancer-associated variants of the human DNA MMR genes MSH2, MSH6, MLH1, and PMS2, however the contribution of each genetic variant to disease is not always clear. We were able to characterize distinct functional defects conferred by cancer-associated variants; we tested seven missense mutations in MSH6, and a predicted truncating mutation in MSH2. We found that several mutations have consequences for protein function, while some may be weakly penetrant alleles or may be polymorphisms. These results could be applied to patient management and familial genetic counseling. ^ In addition to examining the consequences of cancer-associated variants, we were interested in using single amino acid mutants to better understand the mechanism of MMR, and to better define disease in the search for treatments. It is known that the DNA MMR recognition complex MSH2-MSH6, binds to DNA lesions and utilizes ATP binding and hydrolysis, coordinated with conformational changes, to relay a signal for repair. However, the mechanism for regulating and coordinating the functions of this asymmetric heterodimeric complex is not fully understood. We used missense mutants to selectively disrupt ATP processing in individual MSH2 and MSH6 subunits in order to test the contribution each subunit makes to the function of the heterodimer. We found that MSH2 together with magnesium is the regulator of MSH2-MSH6 activity. This evidence clearly supports the molecular switch model for MMR.