Dominant negative mutator mutations in the mutS gene of Escherichia coli

Wu, Teresa; Marinus, Martin

doi:10.1128/jb.176.17.5393-5400.1994

Cited by 125 publications

(112 citation statements)

References 43 publications

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“…Second, we compared the survival of GM56 Δada Δogt cells growing in succinate which contained pBR322, or pET derivatives of mutS, mutL and mutH. The pET derivatives were chosen because they do not have the coding region upstream of these genes and because they complement mut deletion mutations on the chromosome in the absence of inducer [35,36]. There was no significant difference in the survival of these strains (data not shown) indicating that alteration in the levels of MutS, MutL and MutH cannot be the explanation for the results shown in Fig.…”

Section: Mmr-induced Dsbs In Cells With a Reduced Number Of Replicatimentioning

confidence: 99%

DNA mismatch repair-induced double-strand breaks

2008

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Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis, that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB (Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose-and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB (Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O 6 -meG base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB (Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.

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Section: Mmr-induced Dsbs In Cells With a Reduced Number Of Replicatimentioning

confidence: 99%

DNA mismatch repair-induced double-strand breaks

2008

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“…For studies in Saccharomyces cerevisiae, the strains, plasmids, and media have been described previously [16,22]. Plasmids pMQ315, which bears the mutS + gene, has been described previously [23]. pMAKSACA, the suicide replacement vector used to modify the mutS + gene in the genome, has been described previously [24].…”

Section: Strainsmentioning

confidence: 99%

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

et al. 2007

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The major eukaryotic mismatch repair (MMR) pathway requires Msh2-Msh6, which, like E. coli MutS, binds to and participates in repair of the two most common replication errors, single base-base and single base insertion-deletion mismatches. For both types of mismatches, the side chain of E. coli Glu38 in a conserved Phe-X-Glu motif interacts with a mismatched base. The Oε of Glu38 forms a hydrogen bond with either the N7 of purines or the N3 of pyrimidines. We show here that changing E. coli Glu38 to alanine results in nearly complete loss of repair of both single base-base and single base deletion mismatches. In contrast, a yeast strain with alanine replacing homologous Glu339 in Msh6 has nearly normal repair for insertion-deletion and most base-base mismatches, but is defective in repairing base-base mismatches characteristic of oxidative stress, e.g., 8-oxo-G•A mismatches. The results suggest that bacterial MutS and yeast Msh2-Msh6 differ in how they recognize and/or process replication errors involving undamaged bases, and that Glu339 in Msh6 may have a specialized role in repairing mismatches containing oxidized bases.

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“…In addition to their mismatch recognition activity, MutS/Msh proteins also possess an ATPase activity that is essential for DNA repair [4][5][6][7]. ATP binding and hydrolysis appear to modulate the interactions between MutS/Msh and DNA as well as other proteins in the repair pathway; thus, understanding how MutS/Msh proteins utilize ATP is necessary for understanding how they function in DNA mismatch repair.…”

Section: Introductionmentioning

confidence: 99%

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

et al. 2006

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Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S 1 ) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S 2 ) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S 1 -but which of these subunits, S 1 or S 2 , makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S 1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S 2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.

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Dominant negative mutator mutations in the mutS gene of Escherichia coli

Cited by 125 publications

References 43 publications

DNA mismatch repair-induced double-strand breaks

DNA mismatch repair-induced double-strand breaks

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

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