Interactions of Human Mismatch Repair Proteins MutSα and MutLα with Proteins of the ATR-Chk1 Pathway

At clinically relevant doses, chemotherapeutic SN1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutS alpha and MutL alpha. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutS alpha and MutL alpha with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutS alpha with ATR, TopBP1, Claspin, and Chk1 and interaction of MutL alpha with TopBP1 and Claspin. We were unable to detect interaction of MutS alpha or MutL alpha with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutS alpha with ATR, TopBP1, and Chk1 and of MutL alpha with TopBP1. MutS alpha Claspin and MutL alpha-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutL alpha- and MutS alpha-dependent fashion after N-methyl-N'-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.