A human 200-kDa protein binds selectively to DNA fragments containing G.T mismatches.

Jiricny, Josef; Hughes, Melya J.; Corman, Natalie; Rudkin, Brian B.

doi:10.1073/pnas.85.23.8860

Cited by 110 publications

(78 citation statements)

References 19 publications

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“…The electrophoretic mobility-shift assays were performed as described (24), except that each reaction contained 50 fmol of the 34-mer G͞T oligo probe, 10 g of the extract (Fig. 4), and 100 ng of poly d(I⅐C) nonspecific competitor (Amersham Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Marra

D’Atri

Corti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Members of hereditary nonpolyposis colon cancer (HNPCC) familiesharboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2 ؉/؊ lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2 ؉/؊ cell lines. In contrast, hMLH1 ؉/؊ heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2 ؉/؊ colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

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Section: Methodsmentioning

confidence: 99%

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Marra

D’Atri

Corti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…1), it might be expected that both polypeptides contact DNA. However, our earlier experiments showed that only hMSH6 could be cross-linked to mismatch-containing DNA (20,26,27). These latter results invoke the asymmetric nature of the heterodimer and imply that the two subunits play distinct roles during mismatch recognition.…”

Section: Postreplicative Mismatch Repair (Mmr)mentioning

confidence: 99%

“…After 72 h of incubation, the cells were harvested and the total protein extracts were prepared as described (26). The proteins were purified by fast protein liquid chromatography (FPLC) essentially as described (24) except that the heparin-Sepharose fractions containing hMutS␣ were first loaded on a Resource-S FPLC column.…”

Section: Site-directed Mutagenesis and Production Of The Recombinant mentioning

confidence: 99%

Mismatch Recognition and DNA-dependent Stimulation of the ATPase Activity of hMutSα Is Abolished by a Single Mutation in the hMSH6 Subunit

Dufner

Marra

Räschle

et al. 2000

Journal of Biological Chemistry

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The most abundant mismatch binding factor in human cells, hMutS␣, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein.The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine 3 alanine substitution within one such motif, GXFY(X) 5 DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., CameriniOtero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutS␣. The Phe 3 Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutS␣ were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutS␣ not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA. Postreplicative mismatch repair (MMR)1 plays a key role in the maintenance of genomic integrity by eliminating base-base mismatches and insertion-deletion loops (IDLs) that arise in DNA during replication or recombination (1, 2). The repair process consists of four principal steps: (i) mismatch recognition, (ii) repairosome assembly, (iii) degradation of the newly synthesized strand from the strand discrimination signal to and past the mismatch, and (iv) resynthesis of the excised DNA tract (1). The mismatch recognition step is of particular interest; numerous in vitro and in vivo experiments, carried out over the past decade, have shown that the MMR system is capable of addressing a large number of substrates ranging from purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine mismatches to IDLs of one to more than 10 extrahelical nucleotides (3-9). How are these various substrates recognized? In Escherichia coli the role of the mismatch recognition factor is played by a homodimer of the MutS protein, which can initiate the repair of base-base mismatches (5, 10) and IDLs up to four nucleotides (7). In human cells, this function is fulfilled by one of two heterodimers consisting of MutS homologues (MSH): hMutS␣, a heterodimer of hMSH2 and hMSH6 (11-13), which acts in the correction of base-base mismatches and small IDLs (8,12,14), and hMutS␤, a heterodimer of hMSH2 and hMSH3, which addresses principally IDLs (8, 14, 15). The situation in Saccharomyces cerevisiae is similar (16 -19).The highly conserved C termini of MutS homologue proteins contain the ATP binding sites as well as helix-turn-helix motifs (20 -22). As nucleotide binding alters the conformation of the DNA-bound factors, such that they...

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“…The recombinant proteins were produced in Spodoptera frugiperda 9 (Sf9) insect cells according to manufacturer's instructions, and the total protein content was extracted as described. 25 All MSH2 proteins were produced together with MSH6 to give rise to the heterodimeric MutSa (MSH2-MSH6) protein complex, whose both partners are required for active MMR. 26,27 Western blot and coimmunoprecipitation…”

Section: Site-directed Mutagenesis and Expression Of Recombinant Protmentioning

confidence: 99%

Uncertain pathogenicity of MSH2 variants N127S and G322D challenges their classification

Ollila

Đermadi

Greenblatt

et al. 2008

Intl Journal of Cancer

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Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in mismatch repair (MMR) genes. Inherited missense mutations, however, complicate the diagnostics because they do not always cause unambiguous predisposition to cancer. This leads to variable and contradictory interpretations of their pathogenicity. Here, we establish evidence for the functionality of the 2 frequently reported variations, MSH2 N127S and G322D, which have been described both as pathogenic and nonpathogenic in literature and databases. We report the results of 3 different functional analyses characterizing the biochemical properties of these protein variants in vitro. We applied an immunoprecipitation assay to assess the MSH2-MSH6 interaction, a bandshift assay to study mismatch recognition and binding, and a MMR assay for repair efficiency. None of the experiments provided evidence on reduced functionality of these proteins as compared to wild-type MSH2. Our data demonstrate that MSH2 N127S and G322D per se are not sufficient to trigger MMR deficiency. This together with variable clinical phenotypes in the mutation carriers suggest no or only low cancer risk in vivo.

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A human 200-kDa protein binds selectively to DNA fragments containing G.T mismatches.

Cited by 110 publications

References 19 publications

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Mismatch Recognition and DNA-dependent Stimulation of the ATPase Activity of hMutSα Is Abolished by a Single Mutation in the hMSH6 Subunit

Uncertain pathogenicity of MSH2 variants N127S and G322D challenges their classification

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A human 200-kDa protein binds selectively to DNA fragments containing G.T mismatches.

Cited by 110 publications

References 19 publications

Tolerance of human MSH2 +/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 +/− lymphoblastoid cells to the methylating agent temozolomide

Mismatch Recognition and DNA-dependent Stimulation of the ATPase Activity of hMutSα Is Abolished by a Single Mutation in the hMSH6 Subunit

Uncertain pathogenicity of MSH2 variants N127S and G322D challenges their classification

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Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide