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.
Highlights
MutS␣, a heterodimer of MSH2 and MSH6, is essential for DNA mismatch repair
The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors
Mutant human MutS␣ proteins MSH2G674A-MSH6wt and MSH2wt-MSH6T1219D are profiled in a variety of functional assays and as expected fail to support MMR in vitro, they retain mismatch recognition activity
Summary
MutS␣, a heterodimer of MSH2 and MSH6, is essential for DNA mismatch repair. Results: hMsh2G674A-hMSH6wt and hMSH2wt-hMSH6T1219D mutant proteins fail to efficiently license mismatch-provoked, nick-directed excision. Residue Gly-674 is located in the Walker A ATP binding motif within the conserved C-terminal ATPase domain of hMSH2, and Thr-1219 is at the hMSH2hMSH6 heterodimer interface in close proximity to the ABC ATPase “signature motif” of hMSH6 and the P loop of the hMSH2 ATP binding site (Fig. 1) [26] These residues have functional significance in human MMR, as Lynch syndrome alleles encode the MSH6T1219I mutation [27], and a mutant hMSH2 protein having a Gly-674 to Arg substitution is defective for MMR in vitro [28]. Edelmann and co-workers generated two knock-in mouse strains harboring Msh2G674A or Msh6T1217D alleles and found that the mice developed cancer, exhibited microsatellite instability, and yielded embryonic fibroblasts that displayed impaired MMR in vitro Their reports indicated these are separation-of-function alleles in that the MMR response is abrogated, but the apoptotic response to DNA-damaging agents is retained [1, 2]
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