A LexA mutant repressor with a relaxed interdomain linker.
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Bacterial Proteins↗
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The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity.
Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H. The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined. A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E. coli DNA-repair system. Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis. From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor. It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis. Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex.
Antibiotics, Antineoplastic↗
Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12.
The recA gene of E. coli is regulated by a specific repressor, the lexA protein, which binds to an operator in the recA regulatory region. We describe in this paper the isolation and characterization of a mutant thought to carry an operator-constitutive mutation in the recA gene. This mutation has the following properties: 1) It partially suppresses the UV sensitivity of lexA- strains. 2) It maps near the recA gene. 3) It allows constitutive high-level synthesis of recA protein in both lexA- and lexA+ backgrounds. 4) It allows constitutive synthesis of the recA messenger RNA. 5) It is cis-acting. The mutation does not restore induced cellular mutagenesis in a lexA- background. The expression of induced repair and mutagenesis of UV irradiated phage lambda or the regulation of the lexA gene is not affected by the presence of the mutation in either a lexA+ or lexA- strain. These observations confirm other findings that high levels of recA protein synthesis per se is not sufficient for the expression of UV inducible functions and that the lexA protein represses other genes besides the recA gene.
Autoradiography↗
New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.
To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.
Bacterial Proteins↗
One-step cloning system for isolation of bacterial lexA-like genes.
A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.
Bacterial Proteins↗
Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12.
The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein. In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions. DNA sequence analysis identified 20 different mutations. In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA. In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction.
Amino Acid Sequence↗
Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation.
The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.
Adenosine Triphosphate↗
In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage.
Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (IndS) and here called IndS, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these IndS mutant proteins, and of several double mutant proteins containing an IndS mutation and one of several mutations, termed Ind-, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the IndS mutant proteins had a higher maximum rate and a lower apparent pKa than wild-type LexA. Second, the IndS mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of Kcat/Km was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind- mutant protein. We interpret these data to mean that the IndS mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.
Bacterial Proteins↗
Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages.
The SOS hypothesis postulated that the mutator effect on undamaged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage lambda for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA+ and umuDC+ genes. In this paper, we show that (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage lambda, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and phi X174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage lambda and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5'----3', activities of PolI. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.
Bacterial Proteins↗
The amino-terminal domain of LexA repressor is alpha-helical but differs from canonical helix-turn-helix proteins: a two-dimensional 1H NMR study.
The amino-terminal DNA binding domain of LexA repressor consisting of 84 amino acid residues has been studied by two-dimensional 1H NMR. Sequence-specific 1H resonance assignments were made for the first 60 amino acid residues. The secondary structure of this part of the protein contains three alpha-helices in the peptide segments 8-20, 28-35, and 41-54. The last helix has a distortion around residues 47-48. The peptide segment 28-47 shows weak homology with other helix-turn-helix proteins. To investigate the spatial structure of this region of the molecule distance-geometry calculations were performed based on proton-proton distance constraints from nuclear Overhauser effects. The resulting structure shows that the segment 28-47 contains two helices with a loop region between them. The relative orientation of the two helices is similar to that found in helix-turn-helix proteins, but the helices are further apart, with the phenyl ring of Phe-37 located between them. The Brookhaven Protein Data Bank was searched for structurally homologous peptide segments in other proteins. The result of this search was that the two-helical structure of LexA is not more closely related to the canonical helix-turn-helix motif than it is to similar substructures found in other classes of proteins.
Amino Acid Sequence↗
DNA-bound Fos proteins activate transcription in yeast.
We constructed genes encoding the DNA binding region of the bacterial LexA repressor fused to the v-fos and c-fos oncogene products. The resulting LexA-Fos fusion proteins activated transcription in yeast. Transcription activation by these proteins was as strong as transcription activation by proteins native to yeast. LexA-Fos fusion proteins only activated transcription of genes when they were bound to LexA binding sites inserted upstream of those genes. Transcription was activated less strongly by similar proteins in which the DNA binding region of LexA was fused to vMyc and cMyc. Transcription was not activated by native LexA or by proteins containing the DNA binding domain of LexA fused to bacteriophage 434 repressor or yeast MAT alpha 2 protein. These results demonstrate that Fos proteins activate eukaryotic gene expression when they are bound to promoter DNA, and thus suggest that Fos proteins exert some of their effects because they stimulate transcription of cellular genes. Regulation of transcription by Fos and Myc proteins in yeast provides a phenotype that may facilitate genetic analysis of the function of these proteins in higher organisms.
Amino Acid Sequence↗
Nucleotide sequence of the lexA gene of E. coli.
Using a cloned fragment containing the lexA gene of E. coli, the entire nucleotide sequence of the lexA gene has been determined. The probable coding region of the lexA gene contains 606 nucleotide residues and encodes a single protein of 202 amino acids. The initiation site of in vitro transcription of the lexA messenger RNA has been determined by analysis of the 5' nucleotide sequence. Comparison of the DNA sequence of the promoter region of the lexA gene with that of the recA gene reveals the presence of sequences that are common to both. There is some similarity between the amino acid sequences of the lexA and the lambda repressor proteins.
Bacterial Proteins↗
Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.
Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes.
Bacterial Proteins↗
Quantitative evaluation of recA gene expression in Escherichia coli.
A recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds. The RecA protein normally represents 0.02% of total protein. This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation. In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a "super-repressor" of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30 degrees C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.
Bacterial Proteins↗
C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs.
RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.
Adenosine Triphosphate↗
lexA dependent recombination in uvrD strains of Escherichia coli.
Mutation of the uvrD gene of Escherichia coli is associated with an increased capacity for genetic recombination. The hyper-recombination effect is abolished by an additional mutation in lexA that limits synthesis of RecA protein and other gene products regulated by LexA repressor, and is not restored when increased synthesis of RecA protein is facilitated by a recAoc mutation. The viability of uvrD lexA strains is reduced and revertants selected on the basis of improved growth fall into three categories: those that are lexA+, or carry another mutation in lexA that directly suppresses the lexA defect; recA mutants that have lost the capacity for recombination altogether; and a third class which carry a mutation that is not in lexA or recA and which restores the hyper-rec phenotype but does not otherwise suppress the lexA defect. These results indicate that the hyper-recombination effect of a uvrD mutation is an induced response catalysed by RecA protein and at least one other lexA regulated activity.
Bacterial Proteins↗
Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli.
The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli. By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene. The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability. Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda. The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101. beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents. Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction.
Bacterial Proteins↗
Latent ClpX-recognition signals ensure LexA destruction after DNA damage.
The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress.
ATPases Associated with Diverse Cellular Activitie↗