title: Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D).
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.
authors.affiliation: Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Hui
authors.initials: H
authors.lastname: Geng
authors.affiliation: Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut 06459, and.
authors.forename: Miho
authors.initials: M
authors.lastname: Sakato
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Vanessa
authors.initials: V
authors.lastname: DeRocco
authors.affiliation: Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamane
authors.affiliation: Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Chunwei
authors.initials: C
authors.lastname: Du
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Dorothy A
authors.initials: DA
authors.lastname: Erie
authors.affiliation: Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut 06459, and.
authors.forename: Manju
authors.initials: M
authors.lastname: Hingorani
authors.affiliation: Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892,. Electronic address: peggyh@intra.niddk.nih.gov.
authors.forename: Peggy
authors.initials: P
authors.lastname: Hsieh
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D053843:DNA Mismatch Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D051718:MutS Homolog 2 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding
chemical_list: D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D004247:DNA; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein