The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls an SOS regulon.
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Amino Acid Sequence↗
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Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks.
E. coli responds to DNA damage by derepressing the transcription of about 20 genes that make up the SOS pathway. Genetic analyses have shown that SOS induction in response to double-stranded DNA (dsDNA) breaks requires LexA repressor, and the RecA and RecBCD enzymes--proteins best known for their role as initiators of dsDNA break repair and homologous recombination. Here we demonstrate that purified RecA protein, RecBCD enzyme, single-stranded DNA-binding (SSB) protein, and LexA repressor respond to dsDNA breaks in vitro by derepressing transcription from an SOS promoter. Interestingly, derepression is more rapid if the DNA containing the dsDNA break has a chi recombination hot spot (5'-GCTGGTGG-3'), suggesting a novel regulatory role for one of the most overrepresented octamers in the E. coli genome.
Bacterial Proteins↗
Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele.
Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264-->Glu) and recA665 (Tyr264-->His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.
Alleles↗
Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair.
Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, DeltauvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage-resulting in an antimutagenic effect on spontaneous mutation.
Acrolein↗
Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction.
Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response that is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA box properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA box properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as one of the factors modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.
Promoter Regions, Genetic↗
Interaction of Escherichia coli RecA protein with LexA repressor. I. LexA repressor cleavage is competitive with binding of a secondary DNA molecule.
Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively. Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels. The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein. However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites. Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive. The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination.
Adenosine Triphosphate↗
Adaptive mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress.
The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding of phenomena called adaptive mutation in microbes indicates that mutation rates can be elevated in response to stress, producing beneficial and other mutations. We review evidence that, in Escherichia coli, two separate mechanisms of stress-induced genetic change occur that revert a lac frameshift allele allowing growth on lactose medium. First, compensatory frameshift ("point") mutations occur by a mechanism that includes DNA double-strand breaks and (we have suggested) their error-prone repair. Point mutation requires induction of the RpoS-dependent general stress response, and the SOS DNA damage response leading to upregulation of the error-prone DNA polymerase DinB (Pol IV), and occurs during a transient limitation of post-replicative mismatch repair activity. A second mechanism, adaptive amplification, entails amplification of the leaky lac allele to 20-50 tandem repeats. These provide sufficient beta-galactosidase activity for growth, thereby apparently deflecting cells from the point mutation pathway. Unlike point mutation, amplification neither occurs in hypermutating cells nor requires SOS or DinB, but like point mutation, amplification requires the RpoS-dependent stress response. Similar processes are being found in other bacterial systems and yeast. Stress-induced genetic changes may underlie much of microbial evolution, pathogenesis and antibiotic resistance, and also cancer formation, progression and drug resistance.
Adaptation, Biological↗
Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.
The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.
Bacterial Proteins↗
SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli.
Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.
DNA↗
Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.
In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.
Amino Acid Sequence↗
The importance of repairing stalled replication forks.
The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.
Bacteria↗
SOS response as an adaptive response to DNA damage in prokaryotes.
Escherichia coli possesses an elaborate adaptive mechanism called the "SOS response" to cope with various types of DNA damage. More than 20 SOS genes, most of which are known to be involved in the functions that promote the survival of DNA-damaged cells, are induced by treatments that damage DNA or inhibit DNA synthesis. All the SOS genes share similar sequences in the regulatory regions called the "SOS box", to which LexA repressor binds to repress the transcription in the absence of DNA damage. The SOS signal appears to be the single-stranded DNA produced in vicinity of DNA damage, to which RecA protein binds to be activated as a coprotease. The activated RecA promotes autocleavage of LexA protein by allosteric interaction, which activates the latent serine protease activity of LexA. The induced products of the SOS genes repair DNA lesions by various mechanisms, including recombination, excision repair and error-prone repair, and as the consequence, the SOS signal in the cell decreases and the repression of the SOS genes is restored.
Bacterial Proteins↗
Induction of the SOS response increases the efficiency of global nucleotide excision repair of cyclobutane pyrimidine dimers, but not 6-4 photoproducts, in UV-irradiated Escherichia coli.
Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair. In Escherichia coli, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response. We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin. Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively. These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage.
Adenosine Triphosphatases↗
Escherichia coli HU protein suppresses DNA-gyrase-mediated illegitimate recombination and SOS induction.
BACKGROUND: The HU protein is an abundant DNA binding protein of bacteria and is a major constituent of the bacterial nucleoid. HU protein is known to be involved in several fundamental biological functions, including DNA supercoiling, DNA replication, site-specific DNA inversion, and transposition. It is generally thought that a functional relationship exists between HU protein and DNA gyrase. RESULTS: We found that an hupA hupB double mutant displays enhanced spontaneous illegitimate recombination during the formation of lambdabio transducing phage in Escherichia coli. Nucleotide sequence analysis of the resulting transducing phages showed that the E. coli bio and lambda recombination sites did not have any homologous sequence. This mutation also enhanced the spontaneous expression of SOS functions. Furthermore, either overproduced GyrA protein or a temperature-sensitive gyrB mutation suppressed the illegitimate recombination enhanced by the defect of HU protein. CONCLUSION: These results show that the defect of HU induces illegitimate recombination and SOS response, which are probably mediated by DNA gyrase, implying that HU protein plays roles in suppression of illegitimate recombination and SOS response through interaction with DNA gyrase.
Bacterial Proteins↗
From DNA damage to mutation in mammalian cells: a review.
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Animals↗
Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli.
A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.
Amino Acid Sequence↗
Mapping of transcriptional start sites of the cea and cei genes of the ColE7 operon.
Two transcriptional start sites were identified 77 and 78 nucleotides upstream of the translation initiation codon of the colicin E7 gene (ceaE7). The guanosine nucleotide located at the fifth position of the SOS box is probably a universal transcriptional start site of all E group colicins. Major and minor transcripts of the immunity gene (cei) are initiated at the 3' end of the cea gene. Relative to the -10 sequence, CAAAAT, of the major ceiE7 promoter, the corresponding region of the cei gene of other E group colicins has an increased content of guanosine nucleotides. However the -10 sequence of the minor ceiE7 promoter, TATGAT, was found to be conserved in other colicin promoters. The results indicate that the structure of the major promoter of the ceiE7 gene is unique among the E group colicins.
Bacteriocin Plasmids↗
Expression of the Escherichia coli dnaQ (mutD) gene is inducible.
By promoter fusion to the galK gene and comparative S1 analysis we investigated the in vivo regulation of transcription of the dnaQ gene which encodes the epsilon-subunit of the DNA polymerase III holoenzyme carrying the 3'----5' exonucleolytic proofreading function. Induction of a mutagenic stress situation by treatment with the base analogue 2-aminopurine (2-AP) leads to an increase in dnaQ transcription. S1 mapping analysis of the two dnaQ transcripts revealed a differential promoter activation for this 2-AP induced increase in dnaQ transcription. In addition, a similar galK promoter fusion with the dnaN gene coding for the beta-subunit of the DNA polymerase III holoenzyme revealed that dnaN transcription is also 2-AP inducible as judged by galactokinase activity. This is the first evidence for the inducibility of dnaQ gene expression (and possibly of other genes of the DNA polymerase II holoenzyme) and is discussed in relation to DNA repair mechanisms.
2-Aminopurine↗