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


The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result
from replication errors. Msh2G674A or Msh6T1217D 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
MSH2G674A-MSH6wt and MSH2wt-MSH6T1219D 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. MSH2wt-MSH6T1219D fails to couple nucleotide binding and mismatch recognition, whereas MSH2G674A-MSH6wt 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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    ABSTRACT: DNA is constantly under attack by a number of both exogenous and endogenous agents that challenge its integrity. Among the mechanisms that have evolved to counteract this deleterious action, mismatch repair (MMR) has specialized in removing DNA biosynthetic errors that occur when replicating the genome. Malfunction or inactivation of this system results in an increase in spontaneous mutability and a strong predisposition to tumor development. Besides this key corrective role, MMR proteins are involved in other pathways of DNA metabolism such as mitotic and meiotic recombination and processing of oxidative damage. Surprisingly, MMR is also required for certain mutagenic processes. The mutagenic MMR has beneficial consequences contributing to the generation of a vast repertoire of antibodies through class switch recombination and somatic hypermutation processes. However, this non-canonical mutagenic MMR also has detrimental effects; it promotes repeat expansions associated with neuromuscular and neurodegenerative diseases and may contribute to cancer/disease-related aberrant mutations and translocations. The reaction responsible for replication error correction has been the most thoroughly studied and it is the subject to numerous reviews. This review describes briefly the biochemistry of MMR and focuses primarily on the non-canonical MMR activities described in mammals as well as emerging research implicating interplay of MMR and chromatin.
    Full-text · Article · Aug 2014 · Frontiers in Genetics
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    • "The DNA-MMR complex initiates the signaling process to replace the DNA altered region through the action of DNA polymerase δ and DNA ligase I [15] [16] (Figure 1(d)). The mechanism that recruits MMR proteins is ATP dependent [17]. Additionally, the activity of the two MutS dimers at the DNA mismatch site is dependent on interactions with the proliferating cell nuclear antigen (PCNA) [18] [19], which is an important cofactor that participates in both DNA replication and repair mechanisms. "
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    ABSTRACT: The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life ("health-span"). The DNA mismatch repair system (MMR) is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others.
    Full-text · Article · Nov 2012 · Oxidative Medicine and Cellular Longevity
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    ABSTRACT: Binding experiments with alkyl-transfer-active and -inactive mutants of human O6-alkylguanine DNA alkyltransferase (AGT) show that it forms an O6-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with a single-stranded substrate indicate that two or more AGT monomers participate in the rate-limiting step, showing for the first time a functional link between cooperative binding and the repair reaction. Alkyl-transfer kinetics with a duplex substrate suggest that two pathways contribute to the formation of the specific 6mG-complex; one at least first order in AGT, we interpret as direct lesion binding. The second, independent of [AGT], is likely to include AGT transfer from distal sites to the lesion in a relatively slow unimolecular step. We propose that transfer between distal and lesion sites is a critical step in the repair process.
    Full-text · Article · Jul 2012 · Nucleic Acids Research
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