Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

Purification of an SOS repressor from Bacillus subtilis.

We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein. We show that the 23-kDa B. subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B. subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA. In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression. There was no detectable decrease in DNA binding activity in B. subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment. The addition of purified B. subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction. We purified the B. subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose. We show that B. subtilis RecA inactivates the DNA binding activity of the purified B. subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate. By analogy with E. coli, our results indicate that the DNA-binding protein is the repressor of the B. subtilis SOS DNA repair system.     

Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.     

Regulation of the SOS response analyzed by RecA protein amplification.

A split UV light dose procedure was used in Escherichia coli to induce an SOS function, RecA protein amplification, which was measured by an immunoradiometric assay. The SOS system was partially induced after the first UV irradiation, and the inducing effects of subsequent identical UV doses were quantified. Variations in the inducing effects of successive UV doses were related to modulations of the SOS signal level during SOS induction. A reduction in the level of SOS signal was found after 20 min in the wild-type strain, hypothesized to result from negative control of repair functions. A few DNA repair mutants were tested by the same procedure; the uvrA, recF, and umuC genes were involved in SOS induction control, but we found differences in their respective kinetics of expression. On the contrary, in a recB mutant, only a slight effect was obtained on this control.     

Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons

The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.     

Purification of a RecA protein analogue from Bacillus subtilis

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.     

UV induction of LexA independent proteins which could be involved in SOS repair

The SOS response is induced in E coli following treatments that interfere with DNA replication. The response is under the control of the recA and the lexA genes. Strains defective in LexA repressor constitutively express SOS proteins. However, SOS repair does not reach its maximum level in these strains. Instead, an activation of RecA protein and de novo protein synthesis are required for full repair. We have analyzed by 2-dimensional gel electrophoresis the induction of proteins after UV irradiation of lexA(Def) bacteria. Proteins which might participate in SOS repair are induced under these conditions.     

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.     

Duplication Mutation as an Sos Response in Escherichia Coli: Enhanced Duplication Formation by a Constitutively Activated Reca

The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes with the presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system produces a large increase in the frequency of point mutations. To examine the possibility that other types of mutations are induced as part of the SOS response, we have studied the production of tandem duplications. To avoid the complications of indirect effects of the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway. The induction of pathways for both duplications and point mutations shows that the SOS system produces a broad mutagenic response. We have suggested previously that many types of mutations might be induced by severe environmental stress, thereby enhancing genetic variation in an endangered population.     

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.     

A bacterial strain for detecting agents that produce free radical-mediated DNA strand breaks

In an E. coli strain carrying two mutations, one in the dnaC gene involved in initiation of DNA replication and another in the uvrB gene which affects the excision-repair system, it has been shown that the SOS response cannot be induced by UV. This is probably due to the absence of any inducing signal (Salles and Defais, 1984). The capacity to induce the SOS network was followed using RecA protein amplification as a probe. When breaks were produced in DNA, RecA protein induction was restored. We describe here a strain in which both RecA protein and beta-galactosidase from a sfiA::lacZ fusion can be measured simultaneously in the same bacterial extract. In conditions in which no replication proceeds, this strain can be used to detect the ability of chemicals to produce free radical-mediated DNA breaks in vivo.     

Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.