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PubMed1987

Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli.

Overproduction of single-stranded DNA-binding protein (SSB) in Escherichia coli led to a decrease in the basal level of repressor LexA. Expression of the LexA-controlled genes was increased differentially, depending on the affinity of the LexA repressor for each promoter: expression of the recA and sfiA genes was increased 5-fold and 1.5-fold, respectively. Despite only a slight effect on expression of sfiA, which codes for an inhibitor of cell division, bacteria overproducing SSB produced elongated cells. In fact, the effect on cell shape appeared to be essentially independent of the expression of the sfiA and recA genes. Bacteria overproducing SSB were therefore phenotypically similar to bacteria partially starved of thymine, in which filamentation results from both sfiA-dependent and sfiA-recA-independent pathways. These data indicate that excess SSB acts primarily by perturbing DNA replication, thereby favoring gratuitous activation of RecA protein to promote cleavage of LexA protein. When bacteria overproducing SSB were exposed to a DNA-damaging agent such as ultraviolet light or mitomycin C, the recA and sfiA genes were fully induced. Induction of the sfiA gene occurred, however, at higher doses in bacteria overproducing SSB protein than in bacteria with normal levels of SSB. Whereas the efficiency of excision repair was apparently increased by excess SSB, the efficiency of post-replication recombinational repair was reduced as judged by a decrease in the recombination proficiency between a prophage and ultraviolet-irradiated heteroimmune infecting phage. Following induction of ssb+ bacteria with mitomycin C, the cellular content of SSB was slightly increased. These results provide evidence that SSB modulates RecA protein-dependent activities in vivo. It is proposed that SSB favors the formation of short complexes of RecA protein and single-stranded DNA that mediate cleavage of the LexA and lambda repressors, while it delays the formation of long nucleoprotein filaments, thereby slowing down RecA-promoted recombinational events in uninduced as well as in induced bacteria.

Bacterial Proteins↗
PubMed1991

Mutant LexA proteins with an increased rate of in vivo cleavage.

LexA repressor of Escherichia coli is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo. This cleavage reaction can proceed in vitro in the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH. Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in vivo, compared with wild-type LexA. Efficient in vivo cleavage of these mutant proteins also took place without RecA. Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion. These mutations changed Gln-92, which lies near the cleavage site, to tyrosine, phenylalanine, or tryptophan. The latter mutation increased the rate of cleavage approximately 500-fold. These findings imply that the rate of wild-type LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNA-damaging treatments. Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.

Amino Acid Sequence↗
PubMed2001

Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage.

Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.

Alleles↗
PubMed2002

Correct integration mediated by integrase-LexA fusion proteins incorporated into HIV-1.

Fusion of wild-type or truncated integrase to a sequence-specific DNA-binding protein, such as the Escherichia coli LexA repressor, results in an integration bias toward the recognition site of the DNA-binding protein in vitro. Integrase-defective HIV-1 could become integration-competent by supplying the fusion protein in trans. To understand the mechanism of complementation, the virus-host DNA junctions of cells infected with the integrase-LexA containing virus were sequenced. The characteristic hallmarks of wild-type integration were present, a 5'-TG/CA-3' at the ends of the viral sequence and a 5-bp direct repeat in the immediately flanking cellular DNA. Experiments were also carried out to determine the mechanism by which the amino- or carboxy-terminal truncated integrase fused to LexA restored integration to the integrase-mutant viral clone. Complementation experiments using purified fusion proteins in vitro, or viruses encoding a C-terminal truncated integrase and containing various fusion proteins in trans, indicated that the truncated integrase-LexA proteins are inactive per se and they restore integration by forming mixed multimers with the virally encoded mutant integrase. Correct integration of retroviral DNA by the in trans method illustrates the feasibility of introducing integrase fusion proteins into retroviral vectors to achieve site-directed integration without interfering with the attributes of the integration reaction.

Amino Acid Substitution↗
PubMed1983

Control of recA dependent activities in Escherichia coli: a possible role for the recF product.

DNA repair and genetic recombination have been studied in recF143 mutants of Escherichia coli in which expression of inducible, SOS repair activities was altered by additional mutations either in lexA or recA. recF143 and lexA3 appeared to act additively to increase sensitivity to UV irradiation and to reduce recombination proficiency. The recF defect was suppressed in strains carrying the tif-1 allele of recA but not in strains carrying mutations that increased synthesis of recA+ protein or which directly inactivated lexA repressor. These and other data are interpreted to suggest that the recF defect is related to a reduced activity of recA protein. The implications of these findings are discussed in relation to the control of SOS activities and cell division during normal growth.

Bacterial Proteins↗
PubMed1990

The LexA repressor and its isolated amino-terminal domain interact cooperatively with poly[d(A-T)], a contiguous pseudo-operator, but not with random DNA: a circular dichroism study.

The interaction of the entire LexA repressor and its amino-terminal DNA binding domain with poly[d(A-T)] and random DNA has been studied by circular dichroism. Binding of both protein species induces an about 2-fold increase of the positive circular dichroism band at about 270 nm of both polynucleotides, allowing a precise determination of the principal parameters as a function of mono- and divalent salt concentration and pH. Both proteins interact much more strongly (about 2000-fold) with poly[d(A-T)] than with random DNA as expected from the homology with the specific consensus binding site of LexA (CTGTATATATATACAG). For both LexA and its DNA binding domain we find that the interaction with poly[d(A-T)] is cooperative with a cooperativity factor omega of about 50-70 for both proteins over a wide range of solvent conditions, suggesting that the carboxy-terminal domain of LexA is not involved in this type of cooperativity. On the contrary, no cooperativity could be detected for the interaction of the LexA DNA binding domain with a random DNA fragment. The overall binding constant K omega (or simply K in the case of random DNA) depends strongly on the salt concentration as observed for most protein-DNA interactions, but the behavior of LexA is unusual in that the steepness of this salt dependence (delta log K omega/delta log [NaCl]) is much more pronounced at slightly acidic pH values as compared to that at neutral or slightly alkaline pH. The behavior is not easily understood in terms of the current theories on the electrostatic contribution to protein-DNA interactions on the basis of polyelectrolyte theory. A comparison of the overall binding constant K omega of the entire LexA repressor and its DNA binding domain reveals that LexA binds only 20-50-fold stronger under a wide variety of salt and pH conditions. This result tends to demonstrate further that the additional energy due to the dimerization of LexA via the carboxy-terminal domain should be rather weak as expected from the small dimerization constant of LexA (2 X 10(-4) M-1).

Bacterial Proteins↗
PubMed1986

Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent.

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.

Bacterial Proteins↗
PubMed1995

Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase.

Agents that damage DNA in Escherichia coli or interfere with its replication induce DNA repair and mutagenesis via the SOS response. This well-known activity is regulated by the RecA protein and the LexA repressor. Following repair or bypass of the DNA lesion, the cell returns to its resting state by a largely unknown process. We found that 2-keto-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase; EC 4.1.3.16) is necessary for the recovery of respiration and that it is regulated by the SOS response. This protein was induced by DNA-damaging agents. Induction required RecA activation. When the LexA regulon was repressed, activation of RecA was not sufficient for induction, indicating the requirement for an additional protein under LexA control. Finally, a mutant in the corresponding hga gene was UV sensitive. 2-Keto-4-hydroxyglutarate aldolase also plays a role in respiratory metabolic pathways, which suggests a mechanism for respiration resumption during the termination of the SOS response.

Base Sequence↗
PubMed1988

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.

Bacterial Proteins↗
PubMed1985

Role of Escherichia coli RecBC enzyme in SOS induction.

Induction of the SOS genes is required for efficient repair of damaged DNA in Escherichia coli. SOS induction by nalidixic acid or oxolinic acid, two inhibitors of DNA gyrase, requires the RecBC enzyme of E. coli. We report here that the nuclease activity of RecBC enzyme is not needed for SOS induction by these agents. We suggest that the unwinding activity of RecBC enzyme produces single-stranded DNA which activates the RecA protein to stimulate LexA repressor cleavage and SOS induction.

DNA Repair↗
PubMed1991

Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis.

An experimental system was used in which His+ mutations induced by ultraviolet light (UV) arise from non-photo-reversible photoproducts whereas lethality is largely determined by photoreversible photoproducts. By exposing a strain with a deletion through recA to light immediately after UV, it was possible to examine mutagenesis under conditions where survival was not significantly different from 100%. No UV mutagenesis was seen in the absence of RecA protein even though the rest of the SOS system was fully expressed due to the presence of a defective LexA repressor and the active carboxy-terminal fragment of UmuD was present as a result of an engineered plasmid-borne gene. We conclude that RecA protein has a third essential function if UV mutagenesis is to be detected in excision-deficient-bacteria. Another experiment showed that in exerting this function RecA protein does not need activation by pyrimidine dimers elsewhere on the genome, in contrast to its protein-cleavage mediation functions with LexA and UmuD proteins. RecA1730 protein blocked UV mutagenesis unless delayed photoreversal was given showing that the third function of RecA protein is not in the misincorporation step. It is therefore most likely to be in the bypass step where UmuD' and UmuC are postulated to act, although the possibility cannot be excluded that RecA protein is required for some other survival function distinct from translesion synthesis.

Alleles↗
PubMed1988

Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates.

To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E. S. Tessman and P. Peterson, J. Bacteriol. 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro. In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition. We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor. The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP. These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein. With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo. The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition.

Adenosine Triphosphate↗
PubMedDate not supplied

Control of the SOS regulatory system by the level of RecA protease.

The SOS regulatory system of E. coli controls the cellular response to DNA damage and other treatments which interfere with normal DNA replication. This system can exist in two states--a repressed state, in which a set of genes (SOS genes) is repressed by the LexA repressor; and an induced state, in which the RecA protein is activated as a specific protease which cleaves LexA repressor, leading to derepression of the SOS genes. This article reviews evidence that the state of the SOS regulatory system is controlled by the level of RecA protease activity. This level is controlled in turn by a reversible activation by one or more cofactors. In vitro studies indicate that ATP or dATP and single-stranded polynucleotide are both required to activate the protease; the identity of the in vivo cofactors ("inducing signals") is not yet certain. New experiments are also described which characterize the in vivo cleavage of LexA repressor. These data support the model that the level of RecA protease controls the state of the regulatory system.

Bacterial Proteins↗
PubMed1984

Autodigestion of lexA and phage lambda repressors.

Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli. In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction. I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein. This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reaction. Several lines of evidence argued that it was not due to a contaminating protease activity. Autodigestion was stimulated by alkaline pH. It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+. The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular. Purified phage lambda repressor also broke down under similar conditions to yield products like those resulting from recA protein action. Phage lambda repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo. This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway. These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease. The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.

Amino Acid Sequence↗
PubMed1988

LexA repressor induces operator-dependent DNA bending.

LexA, the repressor of the SOS system in Escherichia coli induces a substantial DNA bending upon interaction with the operator of the caa gene, which codes for the bacterial toxin colicin A. Analysis by gel electrophoresis of a family of DNA fragments of identical length, but bearing the caa operator at different positions, shows that DNA bending occurs close to or within the operator sequence upon LexA binding. In contrast, the interaction of LexA with the recA operator induces no detectable bending on 5% polyacrylamide gels. This difference between the two operators is likely to be due to an intrinsic bendability of the caa operator related to thymine tracts located on both sides of the operator. Such tracts do not exist in the recA operator. The free DNA fragments harbouring the caa operator show a slight tendency to bend even in the absence of the LexA repressor. The centre of this intrinsic bend is located close to or within the caa operator.

Bacterial Proteins↗
PubMed1984

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA.

The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.

Bacterial Proteins↗
PubMed1986

ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.

Adenosine Triphosphate↗
PubMed1997

Characterization of DinR, the Bacillus subtilis SOS repressor.

In Bacillus subtilis, exposure to DNA damage and the development of natural competence lead to the induction of the SOS regulon. It has been hypothesized that the DinR protein is the cellular repressor of the B. subtilis SOS system due to its homology to the Escherichia coli LexA transcriptional repressor. Indeed, comparison of DinR and its homologs from gram-negative and -positive bacteria revealed conserved structural motifs within the carboxyl-terminal domain that are believed to be important for autocatalysis of the protein. In contrast, regions within the DNA binding domain were conserved only within gram-negative or -positive genera, which possibly explains the differences in the sequence specificities between gram-negative and gram-positive SOS boxes. The hypothesis that DinR is the repressor of the SOS regulon in B. subtilis has been tested through overexpression, purification, and characterization of the DinR protein. Like E. coli LexA, B. subtilis DinR undergoes an autocatalytic reaction at alkaline pH at a siscile Ala91-Gly92 bond. The cleavage reaction can also be mediated in vitro under more physiological conditions by the E. coli RecA protein. By using electrophoretic mobility shift assays, we demonstrated that DinR interacts with the previously characterized SOS box of the B. subtilis recA gene, but not with sequences containing single base pair mutations within the SOS box. Together, these observations strongly suggest that DinR is the repressor of the SOS regulon in B. subtilis.

Amino Acid Sequence↗