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1.
Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant 总被引:5,自引:0,他引:5
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Marinus MG 《Journal of bacteriology》2000,182(2):463-468
Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents. 相似文献
2.
The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells. 相似文献
3.
Azide-induced mutagenesis was investigated in Salmonella typhimurium and Escherichia coli. Azide was highly effective in inducing mutation in uvrB, uvrB recA and uvrB recB mutants of S. typhimurium. The mutagenic effect of azide was also observed in uvrA lexA mutants of E. coli K12 and E. coli B/r. These results suggest that azide-induced mutagenesis is due to mis-replication of DNA. 相似文献
4.
recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12. 总被引:1,自引:0,他引:1
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The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein. 相似文献
5.
lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli
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The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains. 相似文献
6.
7.
DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background. 相似文献
8.
Characterization of a dam Mutant of Serratia marcescens and Nucleotide Sequence of the dam Region
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Tammo Ostendorf Peter Cherepanov Johann de Vries Wilfried Wackernagel 《Journal of bacteriology》1999,181(13):3880-3885
The DNA of Serratia marcescens has N6-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from S. marcescens Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (dam) coding for the adenine methyltransferase (Dam enzyme) of S. marcescens was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a dam-13::Tn9 mutant of Escherichia coli. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of E. coli. The dam gene is located between flanking genes which are similar to those found to the sides of the E. coli dam gene. The results of complementation studies indicated that like Dam of E. coli and unlike Dam of Vibrio cholerae, the Dam enzyme of S. marcescens plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors. 相似文献
9.
Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors. 相似文献
10.
Specific A/G-to-C.G mismatch repair in Salmonella typhimurium LT2 requires the mutB gene product. 总被引:5,自引:3,他引:2
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An assay has been developed that permits analysis of repair of A/G mismatches to C.G base pairs in cell extracts of Salmonella typhimurium LT2. This A/G mismatch repair is independent of ATP, dam methylation, and mutS gene function. The gene product of mutB has been shown to be involved in the dam-independent pathway through the in vitro assay. Moreover, specific DNA-protein complexes and an endonuclease can be detected in S. typhimurium extracts by using DNA fragments containing an A/G mismatch. These activities are not observed with substrates which have a T/G mismatch or no mismatch. The S. typhimurium endonuclease, like the A/G endonuclease found in Escherichia coli (A-L. Lu and D.-Y. Chang, Cell 54:805-812, 1988), makes incisions at the first phosphodiester bond 3' to and the the second phosphodiester bond 5' to the dA of the A/G mismatch. No incision site was detected on the other DNA strand. Extracts prepared from mutB mutants cannot form A/G mismatch-specific DNA-protein complexes and do not contain the A/G endonuclease activity. Thus the A/G mismatch specific binding and nicking activities are probably involved in the A/G mismatch repair pathway. Preliminary analysis of the mutational spectrum of the mutB strain has indicated that this mutator allele causes an increase in C.G-to-A.T transversions without affecting the frequencies of other transversion or transition events. In addition, the mutB gene has been mapped to the 64-min region of the S. typhimurium chromosome. Together, this biochemical and genetic evidence suggests that the mutB gene product of S. typhimurium is the homolog of the E. coli micA (and/or mutY) gene product. 相似文献
11.
Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. 总被引:27,自引:4,他引:27
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In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin. 相似文献
12.
UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA+ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate (K. Overbye and P. Margolin, J. Bacteriol. 146:170-178, 1981; K. Overbye, S. M. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Mitomycin C induction of the phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead us to deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability. 相似文献
13.
Escherichia coli dam cells are more susceptible to the cytotoxic action of cisplatin than wildtype. Dam mutS or dam mutL bacteria, however, are resistant to this agent indicating that active mismatch repair sensitizes dam cells to cisplatin toxicity. Genetic data, obtained previously, were consistent with the generation and repair of cisplatin-induced double-strand breaks (DSBs). We measured DSB formation in temperature-sensitive dam recB mutants, after exposure to cisplatin, using pulse field gel electrophoresis and observed an increase in linear 100-300 kb DNA fragments corresponding to approximately 15-45 double strand breaks per genome. The formation of these DSBs was temperature and dose-dependent and was decreased in recBC bacteria at the permissive temperature or in dam(+) or mutS control strains. There was a three-fold increase in circa 2 mb linear chromosomal fragments in dam recBC strains at the non-permissive temperature compared to recBC alone. We show that dam priA strains are not viable suggesting that DSB formation is dependent on DNA replication restart. The sensitivity of priA mutants to cisplatin is also consistent with this conclusion. 相似文献
14.
Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities. 总被引:1,自引:0,他引:1
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B Clerch X Garriga E Torrents C M Rosales M Llagostera 《Journal of bacteriology》1996,178(10):2890-2896
Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants. 相似文献
15.
Using a screen that directly assesses transductional proficiency, we have isolated suppressors of recB mutations in Salmonella typhimurium. The alleles of sbcB reported here are phenotypically distinct from those isolated in Escherichia coli in that they restore recombination proficiency (Rec(+)), resistance to ultraviolet light (UV(R)), and mitomycin C resistance (MC(R)) in the absence of an accompanying sbcCD mutation. In addition the sbcB alleles reported here are co-dominant to sbcB(+). We have also isolated insertion and deletion mutants of the sbcB locus. These null mutations suppress only the UV(S) phenotype of recB mutants. We have also isolated sbcCD mutations, which map near proC. These sbcCD mutations increase the viability, recombination proficiency and MC(R) of both the transductional recombination suppressors (sbcB1 & sbcB6) and the sbcB null mutations. S. typhimurium recB sbcB1 sbcCD8 strains are 15-fold more recombination proficient than wild-type strains. The increase in transductants in these strains is accompanied by a loss of abortive transductants suggesting that these fragments are accessible to the mutant recombination apparatus. Using tandem duplications, we have constructed sbcB merodiploids and found that, in a recB mutant sbcCD(+) genetic background, the sbcB(+) allele is dominant to sbcB1 for transductional recombination but co-dominant for UV(R) and MC(R). However, in a recB sbcCD8 genetic background, the sbcB1 mutation is co-dominant to sbcB(+) for all phenotypes. Our results lead us to suggest that the SbcB and SbcCD proteins have roles in RecBCD-dependent recombination. 相似文献
16.
Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed (mut H,L,S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10(3)-fold. The interspecific recombination in mutS or mutL mutants is only approximately 10-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased approximately 10(3)-fold in a recC mutant. 相似文献
17.
The role of recombination genes in the processing of DNA damaged by methlymethane sulfonate (MMS) was examined in an xth nth nfo strain of Escherichia coli K-12. Introduction of a recQ mutation did not increase the cell's sensitivity to MMS treatment. The presence of recF, recJ or recN mutation slightly increased the cell's sensitivity to MMS treatment. The introduction of recA or recB mutation into the cells led to inviability. Taken together, we suggest that replication of DNA containing apurinic/apyrimidinic (AP) sites in vivo will lead to the formation of secondary lesions. The repair of these secondary lesions requires the function of recA and recB genes, but does not appear to require recF, recJ, recQ or recN genes. 相似文献
18.
Enterobacterial GATC-specific DNA adenine methyltransferase (Dam) plays an essential role in regulation of DNA replication, methyl-directed mismatch repair, transposition and gene expression. In Salmonella typhimurium it has been shown to directly control virulence. In this paper we report cloning and expression of the dam gene from the Shiga toxin-producing VT2-Sa prophage of enterohemorrhagic Escherichia coli O157. Comparisons of the predicted amino acid sequence indicates that Dam methyltransferases of E. coli phages VT2-Sa, 933W, T1 and Haemophilus influenzae phage HP1 make up a separate subgroup of adenine-N6 methyltransferases. These proteins are similar to the gamma subfamily of amino-methyltransferases in respect to the linear order of sequence motifs and the presence of the hallmark "NPPY" tetrapeptide. However, they apparently lack an autonomous target-recognizing domain at the C-terminus of the catalytic domain and therefore we propose to dub them as a "mini-gamma" subfamily. 相似文献
19.
Mismatch Repair Mutations of ESCHERICHIA COLI K12 Enhance Transposon Excision 总被引:12,自引:0,他引:12
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Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms. 相似文献
20.
Studies on the genotoxicity of endosulfan in bacterial systems 总被引:15,自引:0,他引:15
Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage. 相似文献