scholarly journals Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Hongbo Liu ◽  
Stephen R Hewitt ◽  
John B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Uv Mutagenesis ◽  
Repair Protein ◽  
Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

scholarly journals DNA structures generated during recombination initiated by mismatch repair of UV-irradiated nonreplicating phage DNA in Escherichia coli: requirements for helicase, exonucleases, and RecF and RecBCD functions.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1175-1186
Author(s):  
W Y Feng ◽  
J B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Repair System ◽  
Lambda Phage ◽  
Dna Structures ◽  
Phage Dna ◽  
Mismatch Repair System ◽  
Recombination Pathway

Abstract During infection of homoimmune Escherichia coli lysogens ("repressed infections"), undamaged nonreplicating lambda phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD-, ExoI-, ExoVII-, or Rec(J-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut+ UvrD+ Exo+) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.

scholarly journals Spontaneous Mutations Occur Near Dam Recognition Sites in a dam  -  Escherichia coli Host

Genetics ◽  
1987 ◽  
Vol 116 (3) ◽  
pp. 343-347
Author(s):  
Margaretha Carraway ◽  
Philip Youderian ◽  
M G Marinus
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Base Pairs ◽  
Dna Mismatch ◽  
Recognition Sites ◽  
Mismatch Repair System ◽  
Phage P22

ABSTRACT The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5′-GATC-3′ sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam  -) strains of E. coli defective in the adenine methylase that recognizes 5′-GATC-3′ sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam  + hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T→G:C transitions that occur within six base pairs of the two 5′-GATC-3′ sequences in the mnt gene. In contrast, the spectrum of mnt  - mutations in a dam  + host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

scholarly journals Mismatch Repair in Escherichia coliCells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ

1998 ◽  
Vol 180 (4) ◽  
pp. 989-993 ◽  
Author(s):  
Reuben S. Harris ◽  
Kimberly J. Ross ◽  
Mary-Jane Lombardo ◽  
Susan M. Rosenberg
Keyword(s):  
Mismatch Repair ◽  
Single Strand ◽  
Mutation Rates ◽  
Repair System ◽  
Exonuclease Activity ◽  
Mismatch Repair System ◽  
Genes Encoding ◽  
Null Mutants

ABSTRACT In vitro, the methyl-directed mismatch repair system ofEscherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

scholarly journals Gene-Targeted Mice Lacking the Trex1 (DNase III) 3′→5′ DNA Exonuclease Develop Inflammatory Myocarditis

2004 ◽  
Vol 24 (15) ◽  
pp. 6719-6727 ◽  
Author(s):  
Masashi Morita ◽  
Gordon Stamp ◽  
Peter Robins ◽  
Anna Dulic ◽  
Ian Rosewell ◽  
...  
Keyword(s):  
Nuclear Dna ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Dna Base Excision Repair ◽  
Dna Base ◽  
Base Excision ◽  
Base Excision Repair System

ABSTRACT TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3′→5′ exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3′ mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1−/− mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3′ termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1−/− mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.

scholarly journals The Escherichia coli Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions

2003 ◽  
Vol 185 (5) ◽  
pp. 1701-1704 ◽  
Author(s):  
Jennifer Wyrzykowski ◽  
Michael R. Volkert
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Dna Breaks ◽  
Base Pairs ◽  
Double Strand Dna Breaks ◽  
Hydrogen Peroxide Treatment ◽  
Mismatch Repair System ◽  
Dna Strands

ABSTRACT A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.

scholarly journals Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function

2003 ◽  
Vol 371 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Barbara QUARESIMA ◽  
Pietro ALIFANO ◽  
Pierfrancesco TASSONE ◽  
Enrico V. AVVEDIMENTO ◽  
Francesco S. COSTANZO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Fold Increase ◽  
Genetic System ◽  
Mutation Rates ◽  
Wild Type ◽  
Mismatched Dna ◽  
Repair Protein

A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

scholarly journals Mutants with Temperature-Sensitive Defects in the Escherichia coli Mismatch Repair System: Sensitivity to Mispairs Generated In Vivo

2005 ◽  
Vol 187 (3) ◽  
pp. 840-846 ◽  
Author(s):  
Esther S. Hong ◽  
Annie Yeung ◽  
Pauline Funchain ◽  
Malgorzata M. Slupska ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Temperature Sensitive ◽  
Repair System ◽  
Wild Type ◽  
System Sensitivity ◽  
Large Numbers ◽  
Mismatch Repair System ◽  
Homeologous Recombination

ABSTRACT We have used direct selections to generate large numbers of mutants of Escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation, mutS60, results in almost normal levels of spontaneous mutations at 37°C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in mutS at 43°C. However, at 37°C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homeologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of mutD (dnaQ), mutC, and ndk. Strains with mutS60 can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with ndk, lacking nucleotide diphosphate kinase, are striking. An ndk mutS60 strain yields four to five times the level of mutations seen in a full knockout of mutS. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.

scholarly journals Saturation of DNA Mismatch Repair and Error Catastrophe by a Base Analogue in Escherichia coli

Genetics ◽  
2002 ◽  
Vol 161 (4) ◽  
pp. 1363-1371
Author(s):  
Kazuo Negishi ◽  
David Loakes ◽  
Roel M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Repair System ◽  
Mutagenic Action ◽  
Wild Type ◽  
Dna Mismatch ◽  
Cell Counts ◽  
Error Catastrophe ◽  
Mismatch Repair System

Abstract Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G · C → A · T and A · T → G · C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL+ gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.

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