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PubMed1991

LexA protein of Escherichia coli represses expression of the Tn5 transposase gene.

The LexA protein of Escherichia coli represses expression of a variety of genes that, by definition, constitute the SOS regulon. Genetic evidence suggests that Tn5 transposition is also regulated by the product of the lexA gene (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). We now show that the LexA protein represses expression of the tnp gene, located in the IS50R component of Tn5, which encodes a transposase, and that LexA does not repress expression of the IS50R inh gene, which encodes an inhibitor of transposition. Elimination of LexA resulted in increased expression of the tnp gene by a factor of 2.7 +/- 0.4, as indicated by the activity of a lacZ gene fused to the tnp gene. LexA protein retarded the electrophoretic movement of a 101-bp segment of IS50R DNA that contained a putative LexA protein-binding site in the tnp promoter; the interaction between the LexA repressor and the promoter region of the tnp gene appears to be relatively weak. These features show that the IS50R tnp gene is a member of the SOS regulon.

Bacterial Proteins↗

PubMed1999

The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.

Amino Acid Sequence↗

PubMed2005

Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions.

ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.

Colicins↗

PubMed2001

The use of radiation-induced bacterial promoters in anaerobic conditions: a means to control gene expression in clostridium-mediated therapy for cancer.

Nuyts, S., Van Mellaert, L., Theys, J., Landuyt, W., Lambin, P. and Anné, J. The Use of Radiation-Induced Bacterial Promoters in Anaerobic Conditions: A Means to Control Gene Expression in Clostridium-Mediated Therapy for Cancer. Radiat. Res. 155, 716-723 (2001). Apathogenic clostridia, which have been genetically engineered to express therapeutic genes, will specifically target hypoxic and necrotic regions in tumors. This specificity can be improved further if the expression of these genes is controlled by a radiation-induced promoter, leading to spatial and temporal control of gene expression. We isolated two radiation-inducible genes of the SOS repair system of Clostridium. Northern blot experiments confirmed radiation activation of the recA and recN genes at a dose of 2 Gy. The promoter region of these genes was isolated and used to regulate expression of the lacZ gene under anaerobic conditions. For the recA promoter, a significant increase of beta-galactosidase activity of 20-30% was seen after 2 Gy irradiation. The recN promoter did not show a significant induction and had a 50-100 times lower basal expression. Treatment of the recombinant clostridial cultures with the cytostatic agent mitomycin C also resulted in a significant increase of beta-galactosidase activity that was under the control of recA or recN promoter. Oxygen does not appear to be necessary in the activation of the SOS repair system by irradiation as tested with Escherichia coli since recA-deficient and recA-containing strains showed similar survival after treatment with UV and ionizing radiation in the presence or absence of oxygen.

Anaerobiosis↗

PubMed1996

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

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.

Bacterial Proteins↗

PubMed2002

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.

Anti-Bacterial Agents↗

PubMed1999

SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions.

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Base Sequence↗

PubMed2006

Induction of competence regulons as a general response to stress in gram-positive bacteria.

Bacterial transformation, a programmed mechanism for genetic exchange originally discovered in Streptococcus pneumoniae, is widespread in bacteria. It is based on the uptake and integration of exogenous DNA into the recipient genome. This review examines whether induction of competence for genetic transformation is a general response to stress in gram-positive bacteria. It compares data obtained with bacteria chosen for their different lifestyles, the soil-dweller Bacillus subtilis and the major human pathogen S. pneumoniae. The review focuses on the relationship between competence and other global responses in B. subtilis, as well as on recent evidence for competence induction in response to DNA damage or antibiotics and for the ability of S. pneumoniae to use competence as a substitute for SOS. This comparison reveals that the two species use different fitness-enhancing strategies in response to stress conditions. Whereas B. subtilis combines competence and SOS induction, S. pneumoniae relies only on competence to generate genetic diversity through transformation.

Bacillus subtilis↗

PubMed1996

Structure of the UmuD' protein and its regulation in response to DNA damage.

For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response'. For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis). Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD'. To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD' mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response.

Amino Acid Sequence↗

PubMed2001

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli.

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Catalysis↗

PubMed1995

A mechanism for induction of the SOS response in E. coli: insights into the regulation of reversible protein polymerization in vivo.

During normal DNA replication, RecA, the principal recombinational repair enzyme of E. coli, cannot assemble its filament on SSB-bound single-stranded DNA at the replication forks. This behavior is paralleled in vitro, where at low Mg2+ concentrations RecA can not polymerize on SSB-bound single-stranded DNA. Inhibition of DNA replication in vivo renders RecA able to polymerize on SSB-bound single-stranded DNA and to activate the SOS response. Although the mechanism of SOS induction is still obscure, abundant in vitro observations indicate that RecA filament formation on SSB-bound single-stranded DNA is facilitated at elevated concentrations of ATP, Mg2+ and spermidine. It is proposed here that inhibition of DNA synthesis in vivo leads to a similar accumulation of ATP and its counter-ions, Mg2+ and spermidine, resulting ultimately in SOS induction. When DNA synthesis is restored, the concentration of ATP, Mg2+ and spermidine returns to normal levels, favoring RecA depolymerization. On the basis of the known structure of RecA, a mechanism for reversible RecA polymerization is presented. In a RecA polymer, the monomers are known to interact with each other primarily through hydrophobic, oppositely charged surfaces. In conditions suboptimal for polymerization, these hydrophobic surfaces of the monomers are possibly masked by electrostatic interactions with other, oppositely charged domains of the monomers. There are known recombinational repair proteins whose specific functions are likely to assist in RecA polymerization or depolymerization. Features of reversible polymerization of eukaryotic proteins tubulin and actin are consistent with the possibility that RecA exploits a general principle for the regulation of reversible protein polymerization.

Actins↗

PubMed2004

Effect of deletion of SOS-induced polymerases, pol II, IV, and V, on spontaneous mutagenesis in Escherichia coli mutD5.

The E. coli dnaQ gene encodes the epsilon subunit of DNA polymerase III (pol III) responsible for the proofreading activity of this polymerase. The mutD5 mutant of dnaQ chronically expresses the SOS response and exhibits a mutator phenotype. In this study we have constructed a set of E. coli AB1157 mutD5 derivatives deleted in genes encoding SOS-induced DNA polymerases, pol II, pol IV, and pol V, and estimated the frequency and specificity of spontaneous argE3-->Arg(+) reversion in exponentially growing and stationary-phase cells of these strains. We found that pol II exerts a profound effect on the specificity of spontaneous mutation in exponentially growing cells. Analysis of growth-dependent Arg(+) revertants in mutD5 polB(+) strains revealed that Arg(+) revertants were due to tRNA suppressor formation, whereas those in mutD5 DeltapolB strains arose by back mutation at the argE3 ochre site. In stationary-phase bacteria, Arg(+)revertants arose mainly by back mutation, regardless of whether they were proficient or deficient in pol II. Our results also indicate that in a mutD5 background, the absence of pol II led to increased frequency of Arg(+) growth-dependent revertants, whereas the lack of pol V caused its dramatic decrease, especially in mutD5 DeltaumuDC and mutD5 DeltaumuDC DeltapolB strains. In contrast, the rate of stationary-phase Arg(+)revertants increased in the absence of pol IV in the mutD5 DeltadinB strain. We postulate that the proofreading activity of pol II excises DNA lesions in exponentially growing cells, whereas pol V and pol IV are more active in stationary-phase cultures.

DNA Polymerase III↗

PubMed1995

Escherichia coli RecG and RecA proteins in R-loop formation.

Escherichia coli rnhA mutants devoid of RNase HI exhibit constitutive stable DNA replication, cSDR, which is thought to be initiated from R-loops stabilized in the absence of RNase HI. We found that a combination of an rnhA and a recG mutation is lethal to the cell. recG mutations that inactivate the helicase activity of RecG protein and inhibit reverse branch migration of Holliday junctions impart phenotypes resembling those of rnhA mutants. Thus, recG mutants display cSDR activity, and recG polA double mutants are inviable as are rnhA polA double mutants. These results suggest that the RecG helicase has a role in preventing R-loop formation. A model that R-loops are formed by assimilation of RNA transcripts into the duplex DNA is discussed. The model further postulates that RecA protein catalyzes this assimilation reaction and that RecG protein counteracts RecA in this reaction, resolves R-loops by its helicase activity, or does both.

Bacterial Proteins↗

PubMed1995

A modified SOS-Chromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction.

A modified SOS-Chromotest bioassay using a chromogenic pad (pad procedure) was developed to test for genotoxicity in sediments directly without extraction. This test is based on the de novo synthesis of beta-galactosidase enzyme by a genetically-engineered E. coli strain PQ37. In the bioassay, an exponential growth phase antibiotic-containing culture of the test bacterium is introduced into a series of tubes with the first tube containing 0.1 gram of sediment. Serial dilutions are then made and the tubes of sediment plus bacterial culture are incubated at 37 degrees C for four hours, followed by placing a drop of each mixture on a chromogenic pad and additional incubation for 20 hours at 37 degrees C. The solid particulates are then washed off with tap water and positive (genotoxic) activity is noted by the presence of a distinctive blue colour on the pad. The SOS-Chromotest pad procedure may be best used as a relative measure of genotoxicity by comparing results to a reference sample. In addition it can also determine sediment cytotoxicity by comparing samples spiked with a genotoxic standard (i.e., 4-nitroquinoline-N-oxide). Preliminary results suggest that this new bioassay is highly sensitive, consistent and discriminating.

4-Nitroquinoline-1-oxide↗

PubMed1997

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway.

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD+ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.

Adenosine Triphosphatases↗

PubMed2004

Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.

AlkB repairs 1-alkyladenine and 3-methylcytosine lesions in DNA by directly reversing the base damage. Although repair studies with randomly alkylated substrates have been performed, the miscoding nature of these and related individually alkylated bases and the suppression of mutagenesis by AlkB within cells have not yet been explored. Here, we address the miscoding potential of 1-methyldeoxyadenosine (m1A), 3-methyldeoxycytidine (m3C), 3-ethyldeoxycytidine (e3C), 1-methyldeoxyguanosine (m1G), and 3-methyldeoxythymidine (m3T) by synthesizing single-stranded vectors containing each alkylated base, followed by vector passage through Escherichia coli. In SOS(-), AlkB-deficient cells, m1A was only 1% mutagenic; however, m3C and e3C were 30% mutagenic, rising to 70% in SOS(+) cells. In contrast, the mutagenicity of m1G and m3T in AlkB(-) cells dropped slightly when SOS polymerases were expressed (m1G from 80% to 66% and m3T from 60% to 53%). Mutagenicity was abrogated for m1A, m3C, and e3C in wild-type (AlkB(+)) cells, whereas m3T mutagenicity was only partially reduced. Remarkably, m1G mutagenicity was also eliminated in AlkB(+) cells, establishing it as a natural AlkB substrate. All lesions were blocks to replication in AlkB-deficient cells. The m1A, m3C, and e3C blockades were completely removed in wild-type cells; the m1G blockade was partially removed and that for m3T was unaffected by the presence of AlkB. All lesions demonstrated enhanced bypass when SOS polymerases were induced. This work provides direct evidence that AlkB suppresses both genotoxicity and mutagenesis by physiologically realistic low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damage in vivo.

Adenine↗

PubMed1998

The Tum protein of coliphage 186 is an antirepressor.

The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, pR and pB, and thereby bring about lytic development. In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.

Bacterial Proteins↗

PubMed1988

Selection of DNA binding sites by regulatory proteins: the LexA protein and the arginine repressor use different strategies for functional specificity.

The DNA sequences in the operator sites of the arginine regulon and of the SOS regulon have been subject to a statistical analysis. A quantitative correlation is found between the statistics of sequence choice and the activity at individual operator sites in both systems, as expected from theoretical considerations [Berg & von Hippel, J.Mol.Biol. (1987) 193, 723-750]. Based on these correlations it is possible to predict the effect of various sequence mutations. There is a significant difference in the slopes of the correlation lines between sequence and activity for the two systems. From this difference it can be expected that individual point mutations in the ARG boxes will have a much smaller effect on activity than similar changes in the SOS boxes. This difference may be related to a strong cooperative activity at tandem ARG boxes while the binding at SOS boxes appears to be mostly noncooperative.

Arginine↗