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At least 235 records · Page 13Linked to original sources
PubMed1999

Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7.

The recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no. 7 recA promoter region with the recA promoter sequences from other alpha-Proteobacteria revealed the presence of the conserved sequence GAACA-N6-G(A/T)AC. Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R. palustris recA gene, confirming that this sequence is the SOS box of R. palustris and probably plays the same role in other alpha-Proteobacteria.

Amino Acid Sequence↗
PubMed2006

The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites.

Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species.

Bacterial Proteins↗
PubMed1997

Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity.

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.

DNA Polymerase II↗
PubMed2006

Genetic evidence for the requirement of RecA loading activity in SOS induction after UV irradiation in Escherichia coli.

The SOS response in Escherichia coli results in the coordinately induced expression of more than 40 genes which occurs when cells are treated with DNA-damaging agents. This response is dependent on RecA (coprotease), LexA (repressor), and the presence of single-stranded DNA (ssDNA). A prerequisite for SOS induction is the formation of a RecA-ssDNA filament. Depending on the DNA substrate, the RecA-ssDNA filament is produced by either RecBCD, RecFOR, or a hybrid recombination mechanism with specific enzyme activities, including helicase, exonuclease, and RecA loading. In this study we examined the role of RecA loading activity in SOS induction after UV irradiation. We performed a genetic analysis of SOS induction in strains with a mutation which eliminates RecA loading activity in the RecBCD enzyme (recB1080 allele). We found that RecA loading activity is essential for SOS induction. In the recB1080 mutant RecQ helicase is not important, whereas RecJ nuclease slightly decreases SOS induction after UV irradiation. In addition, we found that the recB1080 mutant exhibited constitutive expression of the SOS regulon. Surprisingly, this constitutive SOS expression was dependent on the RecJ protein but not on RecFOR, implying that there is a different mechanism of RecA loading for constitutive SOS expression.

Escherichia coli↗
PubMed2004

Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation.

The cyanobacterial genes lexA, recA and ruvB were analysed in Synechocystis PCC6803, which is shown here to be more radiation resistant than the other unicellular model strain Synechococcus PCC7942. We found that cyanobacteria do not have an Escherichia coli-type SOS regulon. The Synechocystis lexA and recA promoters were found to be strong and UV insensitive, unlike the ruvB promoter, which is weak and UV-C inducible. Yet, lexA and recA are regulated by UV-C, but the control is negative and occurs at the post-transcriptional level. Two novel conserved elements were characterized in the lexA promoter: (i) an unusually long crucial box 5'-TAAAATTTTGTATCTTTT-3' (-64, -47); and (ii) a negatively acting motif 5'-TAT GAT-3' (-42, -37). These elements were not found in the recA promoter, which appeared to be unusually simple in harbouring only a single crucial element (i.e. the canonical -10 box). RuvB, operating in recombination-dependent cellular processes, was found to be dispensable to cell growth, whereas LexA and RecA appeared to be critical to cell viability. Using DNA microarrays, we have identified 57 genes with expression that is altered, at least twofold, in response to LexA depletion. None of these genes is predicted to operate in DNA metabolism, arguing against the involvement of LexA in the regulation of DNA repair. Instead, most of the LexA-responsive genes were known to be involved in carbon assimilation or controlled by carbon availability. Consistently, the growth of the LexA-depleted strain was found to be strongly dependent on the availability of inorganic carbon.

Bacterial Proteins↗
PubMed1997

SOS factors involved in translesion synthesis.

Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon. To study translesion synthesis in vivo, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC+ gene products, whereas elongation from the slipped intermediate is umuDC+ independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.

2-Acetylaminofluorene↗
PubMed2000

The SOS response regulates adaptive mutation.

Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.

Adaptation, Physiological↗
PubMed1998

The Bacillus subtilis DinR binding site: redefinition of the consensus sequence.

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB, dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5' and 3' of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5'-CGAACRNRYGTTYC-3'. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

Amino Acid Sequence↗
PubMed2000

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

Alginates↗
PubMed1996

SOS chromotest results in a broader context: empirical relationships between genotoxic potency, mutagenic potency, and carcinogenic potency.

Environmental monitoring requires that large numbers of samples be processed in a relatively short period of time. While microbioassays facilitate rapid testing, the results are often difficult to interpret in the broader context of human or animal health. Determining the consequences of exposure to genotoxic substances will ultimately require in situ monitoring of exposed organisms. However, it is immediately possible to construct a broad empirical framework within which available microbioassay results can be interpreted. To do this for SOS Chromotest results, we investigated the empirical relationships between SOS genotoxic potency and mutagenic potency (as measured with the Salmonella/microsome assay), as well as between genotoxic potency and carcinogenic potency (as measured using standard, chronic animal bioassays). Strong relationships were identified between; 1) genotoxic potency and mutagenic potency for 268 direct-acting substances (r2=0.76) and 2) genotoxic potency and mutagenic potency for 126 S9-activated substances (r2=0.65). Ordinary least squares regression analyses of the SOS genotoxicity-Salmonella mutagenicity relationship revealed a significant effect of SOS genotoxicity as well as differences in mutagenic potency that can be attributed to the Salmonella strain used to measure mutagenic potency. Analyses of S9-activated substances revealed a significant interaction between the SOS genotoxic potency (SOSIP) effect and the Salmonella strain effect. Two regression models relating SOS genotoxicity and Salmonella mutagenicity were used to predict the mutagenic potency of several industrial effluent extracts previously analyzed for SOS genotoxicity by White et al. [(1996): Environ Mol Mutagen 27:116-139]. Predictions are consistent with published mutagenic potency values for similar industrial waste materials. A consistent relationship was also identified between genotoxic potency and carcinogenic potency for 51 substances. Linear regression analyses revealed an effect of SOS genotoxic potency as well as differences in carcinogenic potency that may be attributable to experimental animal and route of exposure. The correlation between genotoxicity and carcinogenicity was fairly weak (maximum r value = 0.51). Previous studies revealed similar strength of association between Ames mutagenicity and carcinogenicity. Predicted carcinogenic potencies of previously examined genotoxic, industrial effluent extracts are generally low compared to the pure substances included in the data set.

Animals↗
PubMed1994

Accuracy of replication past the T-C (6-4) adduct.

The thymine-cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts. We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS-induced and uninduced cells of a uvr A6 strain of Escherichia coli. Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied. Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions. The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3' C-->T transitions. The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3' C-->T transitions. For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer. The occurrence of the 3' C-->T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction. Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells. Following SOS induction the frequency of bypass increased to 24.5% and 12.5% for the UVC and the Dewar isomers, respectively.

Base Sequence↗
PubMed1993

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli.

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind-) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations oc in the umuDC and umuD'C operons and also used the oc98-recA mutation. The oc1-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutagenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.

Bacterial Proteins↗
PubMed2006

Targeted deletion of the uvrBA operon and biological containment in the industrially important Bacillus licheniformis.

From a Bacillus licheniformis wild type as well as a defined asporogenous derivative, stable UV hypersensitive mutants were generated by targeted deletion of the uvrBA operon, encoding highly conserved key components of the nucleotide excision repair. Comparative studies, which included the respective parental strains, revealed no negative side effects of the deletion, neither on enzyme secretion nor on vegetative propagation. Thus, the uvrBA locus proved to be a useful deletion target for achieving biological containment in this industrially exploited bacterium. In contrast to recA mutants, which also display UV hypersensitivity, further strain development via homologous recombination techniques will be still possible in such uvr mutants.

Bacillus↗
PubMed1991

Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal.

Three models have been proposed for the nature of the SOS-inducing signal in E. coli. One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.

Bacteriophage lambda↗
PubMed1998

Identification of the Rhodobacter sphaeroides SOS box.

Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.

Adenosine Triphosphatases↗
PubMed1990

Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector.

We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E. coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites. Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations. 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction. In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide. At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP. At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP. The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself. In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide.

Bacteriophages↗
PubMed2006

Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene.

Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum lexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the lexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.

Alphaproteobacteria↗
PubMed2005

LexA cleavage is required for CTX prophage induction.

The physiologic conditions and molecular interactions that control phage production have been studied in few temperate phages. We investigated the mechanisms that regulate production of CTXphi, a temperate filamentous phage that infects Vibrio cholerae and encodes cholera toxin. In CTXphi lysogens, the activity of P(rstA), the only CTXphi promoter required for CTX prophage development, is repressed by RstR, the CTXvphi repressor. We found that the V. cholerae SOS response regulates CTXvphi production. The molecular mechanism by which this cellular response to DNA damage controls CTXphi production differs from that by which the E. coli SOS response controls induction of many prophages. UV-stimulated CTXphi production required RecA-dependent autocleavage of LexA, a repressor that controls expression of numerous host DNA repair genes. LexA and RstR both bind to and repress P(rstA). Thus, CTXphi production is controlled by a cellular repressor whose activity is regulated by the cell's response to DNA damage.

Antibiotics, Antineoplastic↗