recA Protein Synthesis Research Articles

Operator-constitutive mutation in the recA gene enhances radiation resistance of Escherichia coli

Plasmid pUC19-recAoc carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli DeltarecA cells, as compared to the wild-type recA+ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recADelta21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAoc effectively suppresses UV sensitivity of the DeltarecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of gamma-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.

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The LexA protein from Deinococcus radiodurans is not involved in RecA induction following gamma irradiation.

The involvement of LexA in induction of RecA was investigated in Deinococcus radiodurans. As in the wild-type strain, an increase in RecA protein synthesis following gamma irradiation was detected in a lexA disruptant, indicating that LexA is not involved in the induction of RecA in D. radiodurans.

Immunological quantification of RecA protein in cell extracts of E. coli after exposure to chemical mutagens or UV radiation.

Increased synthesis of RecA protein is induced in E. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined in E. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). Purified E. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of the RecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5-50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes.

Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli.

Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC. Recovery of DNA synthesis after UV irradiation ("induced replisome reactivation," or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein [Khidhir, M. A., Casaregola, S. & Holland, I. B. (1985) Mol. Gen. Genet. 199, 133-140]. IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants. In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present. IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation. We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein. We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis.

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide.

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant.

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.

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.

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of λ prophage in Dcm + strains but not in Dcm − mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm + or Dcm − strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.

Induction of SOS genes of Escherichia coli by chromium compounds.

The induction of several SOS genes of Escherichia coli such as recA, umuC, and sfiA by hexavalent (K2Cr2O7, K2CrO4, and CrO3) and trivalent (CrCl3, Cr(NO3)3, and (CH3COO)3Cr) compounds of chromium was studied. Induction was measured as beta-galactosidase activity, using lacZ gene fusions under the control region of different SOS genes. The hexavalent chromium forms induced the genes responsible for massive synthesis of RecA protein, error-prone repair, and inhibition of cell division. On the other hand, the trivalent chromium compounds were unable to induce any of the SOS genes tested. Individual assay of hexavalent chromium compounds showed that K2Cr2O7 was a stronger inducing agent of those three SOS genes tested than K2CrO4, which, in turn, was stronger than CrO3. All this data led to the conclusion that hexavalent chromium compounds, but not trivalent, are proficient agents of induction of the SOS system and can produce indirect mutagenesis in Escherichia coli.

The sfiA11 mutation prevents filamentation in a response to cell wall damage only in a recA+ genetic background.

N-alpha-palmitoyl-L-lysyl-L-lysine dihydrochloride ethyl ester (PLL) at sublethal doses causes filamentous growth of E. coli strains except sfiA mutants, which divide normally in its presence. PLL does not elicit the SOS responses as judged by lambda prophage induction, an increase of RecA protein synthesis or induction of the sfiA operon in a sfiA::lacZ fusion strain. Thus, it appears that filamentation caused by PLL is not an SOS function and might be the result of membrane damage by PLL, which is an amphipathic compound and at higher doses causes cell lysis. This indicates that basal levels of the sfiA gene product are sufficient to inhibit cell division in the presence of PLL. We have found further that the phenotype of the sfiA mutation in the presence of PLL requires a recA+ genetic background and does not occur in E. coli recA1 sfiA11, recA13 sfiA11, recA56 sfiA11 and recA441 sfiA11. All these strains, but rec441 sfiA11, however, regain the ability of sfiA11 mutants to divide in the presence of PLL after transformation with the RecA overproducing-plasmid pXO2. This supports the conclusion that the RecA protein positively affects sfiA11-mediated cell division in the presence of the cell membrane damaging compound, PLL. The basal level of the RecA protein in the recA+ sfiA11 strain is sufficient for this process. An increased level due to overproduction from the multicopy plasmid pXO2 exerts the same effect.

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli.

The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvr A6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp+ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp+ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7-2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvr A6 background.

Direct selection of mutations reducing transcription or translation of the recA gene of Escherichia coli with a recA-lacZ protein fusion.

When a recA-lacZ protein fusion was cloned into phage lambda, the resulting transducing phage grew normally on wild-type Escherichia coli, but its growth was severely inhibited in lexA(Def) mutant strains that express recA constitutively at high levels. Mutants of the transducing phage that grew on the lexA(Def) strains were isolated and were found to affect production of the RecA-beta-galactosidase hybrid protein. Most mutants, including a number of nonsense mutants, were phenotypically LacZ-. LacZ+ mutants were also isolated; most of these expressed lower basal and induced levels of beta-galactosidase activity. DNA sequence analysis revealed that some of the LacZ+ mutations were in the recA promoter. One of these was found to prevent induction. Unexpectedly, three of the mutations that reduced expression were located in the recA structural gene, at codons 10, 11, and 12. Further analysis of the codon 10 mutant showed that it most likely affected translation since it had little effect on transcription as measured by beta-galactosidase synthesis from a recA-lacZ operon fusion. This expression defect was not limited to the protein fusion, since the codon 10 mutation also reduced synthesis of RecA protein when present in a complete recA gene. Analysis of the recA DNA sequence in the fusion revealed that each of the mutations at codons 10, 11, and 12 increases the homology between this region of the mRNA and a sequence found at codons 1 to 4. Thus, the secondary structure of the mutant recA mRNAs may be affecting translation.

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC − mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC − cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC − mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC − and wild-type strains.

A loss of uvA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli

We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis. Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau. The induction was blocked by recA and lexA(Ind −) mutations that conferred no mutagenesis on the cell. Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C. The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants.

An endonuclease specific for single-stranded DNA selectively damages the genomic DNA and induces the SOS response.

A plasmid carrying the bacteriophage T7.3 endonuclease gene under the control of the lacUV5 promoter could be maintained in the transcriptionally active state only in recA+ strains. In recA- strains, endonuclease induction resulted in extensive degradation of the genomic DNA and cell death. In sharp contrast, the plasmid DNA remained intact in the supercoiled form. In recA+ strains, the recA protein levels were increased and the SOS functions of the host were activated, as shown by measurements of recA protein synthesis and prophage induction. These results indicate that in normal undisrupted and non-irradiated cells, enzymatic nucleolytic damage can induce the SOS response and can be controlled by the DNA repair system of the host. In addition, the higher sensitivity of the genomic DNA to the single-strand-specific endonuclease relative to the plasmid suggests that the two molecules differ in their physiological states and most likely in their degree of single-stranded content.

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

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

Restoration of RecA protein activity by genetic complementation.

Bacteria carrying either recA430 or recA453-441 mutations are sensitive to UV-irradiation since they amplify the synthesis of RecA protein either poorly or not at all. We show here that, in a recA453-441 (recA430) heterodiploid, UV-resistance and amplification of RecA430 protein were restored, indicating that the cellular level of RecA-associated protease activity was high enough to inactivate LexA repressor. Prophage 434 repressor was also extensively inactivated, whereas RecA430 protein alone cannot cleave this substrate. On the other hand, during growth of the recA453-441(recA430) heterodiploid at 42 degrees C in the presence of adenine, a treatment activating only RecA441 protein, RecA441 protease activity was as high as in a recA441 haploid. In contrast, following this inducing treatment, there was no complementation between RecA441 and RecA+ proteins in a recA453-441(recA+) heterodiploid. These results indicate that multimerization of RecA protein molecules results in a functional interaction that, in some combination between RecA protein subunits, may enhance RecA-associated protease activity.

UV-lysogenic induction of lambda phage in lexA1 mutants of Escherichia coli: kinetics of the process.

Abstract— Although the lex gene has been described until recently as being required for lysogenic induction, both our work and the work of others have reported Λ prophage induction in some lexA mutants. However, the characteristics of the process were not defined. We describe UV induction of prophage in a lexA1 mutant at a slightly lower level and requiring 2 times longer than the wild type. As demonstrated in some work, in cells treated with low levels of rifampicin (RIF) no new synthesis of RecA protein is needed for the prophage induction although the onset of lysis is delayed. We suggest that the lysogenic induction in lexA cells is due to the same mechanism that induces prophage in the wild type cells treated with RIF. That is, the induction is due to the cleavage of Λ represser by the basal RecA protease in the DNA‐single‐strand gap, since RecA protease and monomer represser both have high affinity for this type of DNA. So, LexA protein need not be cleaved for the prophage induction.No Weigle‐reactivation (WR) was detected in the lex mutant even after a long post‐irradiation incubation, suggesting that unlike prophage induction, WR requires LexA protein cleavage.

Survival and induction of recA protein in mitomycin C-treated Escherichia coli rec, lex, or uvr strains.

The capability to synthesize recA protein has been tested for Escherichia coli treated with mitomycin C. recA protein was assayed using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). Mitomycin C-treated wild type E. coli can express recA gene in a similar quantitative fashion, independently of the growth media used in this work; glucose did not inhibit induction of recA protein in cells growing in synthetic media. Wild type E. coli recovering from energy starvation displays a similar qualitative capability to induce the synthesis of recA protein independently of the stage of growth at which the cells are treated with the drug. At midexponential phase, the cells appear to have an enhanced capability to synthesize recA protein. The relationship between survival and capability to synthesize recA protein was explored for E. coli lex, rec, and/or uvr mutants, after treatment with mitomycin C. A good correlation was found, except for a recB mutant and for an ethidium-sensitive strain, both able to produce as much recA protein as the wild type but 100-fold more sensitive to the drug. A similarly satisfactory correlation was found when plotting the survival after UV irradiation versus the capability of synthetizing recA protein with the exception of an uvrA strain and of a lexA strain.