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Biochemical analysis of the human mismatch repair proteins hMutSalpha MSH2(G674A)–MSH6 and MSH2–MSH6(T1219D)

Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2012; 287(13):9777-91. DOI: 10.1074/jbc.M111.316919
Source: PubMed

ABSTRACT The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

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    • "This model is supported by the finding that Msh2-mutant mice carrying a missense mutation Msh2G674A/G674A show less pronounced CTG expansions than wild type mice (Tome et al., 2009). This mutation retains mismatch recognition activity but fails to support MMR in vitro (Lin et al., 2004; Ollila et al., 2008; Geng et al., 2012). In an effort to gain a mechanistic insight into the MMR-dependent instability process, biochemical studies were undertaken. "
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