Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction, Our in vitro MMR assay demonstrated that the dominant-negative hMRE11(452-634) mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 31 excision and 3'-directed MMR activities. Furthermore, the expression of hMRE11(452-634), and to a lesser extent hMRE11(1-634) (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (> 50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants. (c) 2008 Elsevier Inc. All rights reserved.