DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o^c) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o^c) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind⁻) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o^c) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.     

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.     

Characterization of the SOS regulon of Caulobacter crescentus

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN₇GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.     

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.     

Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r.

Summary In Escherichia coli B/r the expression of UV inducible (SOS) functions is under the control of the recA and lexA genes. In this study we have characterized mutants which are altered in their ability to express SOS functions. These mutants were isolated as UV resistant UV nonmutable (Rnm) derivatives of the lexA102 uvrA155 mutant strain WP51. The UV resistance of these Rnm strains is a result of the suppression of lexA102 mediated UV sensitivity. Genetic mapping of rnm mutations shows that the two predominant classes, rnmA and rnmB, map in or very near the lexA and recA genes respectively. rnmA mutations differ from rnmB with respectively recA protein synthesis. rnmA mutations do not restore the ability to express high levels of recA protein after UV treatment whereas rnmB mutations result in constitutive expression of high levels of recA protein. However, both rnmA and rnmB mutant strains inhibit postirradiation DNA degradation. This shows that in rnmA strains, high levels of recA protein are not needed to inhibit postirradiation DNA degradation.The genetic map location and constitutive expression of recA protein synthesis resulting from rnmB mutations suggests that they are operator constitutive mutations of the recA gene. The result that the lexA ⁺ gene is required for the expression of UV mutagenesis in rnmB mutants shows that high levels of recA protein do not circumvent the need for the lexA ⁺ gene product in this process. Thus, while the lexA gene product is required for the induction of recA protein synthesis, lexA must have an additional role in UV induced mutagenesis.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE⁺ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB⁺ compared to its interaction with dnaE⁺. Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB⁺ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE⁺ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.     

Suppression of the E. coli SOS response by dNTP pool changes

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30⁺-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.     

Influence of plasmids carrying the lexA gene on DNA repair and related processes in Escherichia coli K-12

Summary Plasmid pLC44-14 from the Clarke and Carbon collection has been shown to carry the lexA gene. The presence of lexA was demonstrated by complementation of tsl mutants which lie close to lexA on the E. coli K-12 linkage map and are probably in the lexA gene, and by crossing the dominant lexA mutation on to pLC44-14 to produce a recombinant plasmid, pSEl, which gave the host cell the properties of a lexA mutant. The lexA gene has been cloned on to pBR322 (Little, 1980). pJL21, which carries the lexA ⁺ gene, rendered the host cell moderately sensitive to UV light, greatly reduced the extent of Weigle reactivation and mutagenesis of UV-irradiated phage , and inhibited induction of protein X by either UV light or nalidixic acid. A similar plasmid carrying a mutant lexA3 allele produced extreme sensitivity to UV light, reduced recombinant production 10 to 50-fold following Hfr x F^– conjugation crosses, and otherwise mimicked the effects of pJL21. Introduction of an amber mutation into the lexA gene carried by the plasmid greatly reduced the UV-sensitivity of the host, thereby indicating that the extreme sensitivity was due to the mutant lexA gene product. These properties of strains with lexA plasmids are thought to originate from high levels of the lexA protein in the cell due to a large plasmid copy number. This protein, which appears from other studies to regulate negatively the recA gene, may inhibit expression of recA or other DNA repair genes when present in excess amounts in the cell.     

An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.     

Analysis of the expression of the <Emphasis Type="Italic">Rhodobacter sphaeroides lexA</Emphasis> gene

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN₇GAACN₇GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.