Quantitative evaluation of recA gene expression in Escherichia coli

Summary 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°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.Abbreviations Ap ampicillin - Km kanamycin - Cm chloramphenicol - Tc terracycline - Sm streptomycin - Ts thermosensitive - Tr thermoresistant - Nal nalidixic acid - X-Gal 5-bromo-4-chloro-3-indolyl--D-galactoside - mito C mitomycin C - LFT low frequency transducing - HFT high frequency transducing     

Expression of the dnaN and dnaQ genes of Escherichia coli is inducible by mitomycin C

Summary The dnaN and dnaQ genes encode the subunit and the subunit of the DNA polymerase III holoenzyme. Using translational fusions to lacZ we found that DNA damage caused by mitomycin C induces expression of the dnaA and dnaQ genes. This induction was not observed in lexA and recA mutants which block the induction of the SOS response, suggesting a relationship between the mechanism(s) of genetic control of DNA polymerase III holoenzyme and the SOS regulatory network. Nevertheless, there is evidence that the mitomycin C induction of dnaN and dnaQ is not a simple lexA-regulated process, because nalidixic acid (an excellent SOS inducer) does not increase dnaN and dnaQ gene expression, and the time course of induction is abnormally slow.     

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.     

SOS induction by thermosensitive replication mutants of miniF plasmid

Summary MiniF, a 9.3 kb fragment of the dispensable F plasmid, carries genes necessary for its replication and partition as well as for the expression of an SOS signal. The arrest of replication of a thermo-sensitive miniFts at 42°C induced SOS functions such as prophage , sfiA expression, W-reactivation of UV-irradiated phage . Two miniF ts9 and ts17 mutations were located within the KpnI fragment (43.6–46.9) in the minimal oriS replicon. Blocking miniF replication by incBC ⁺ incompatibility genes situated in trans on a second plasmid also induced SOS functions. In contrast, if miniFts17 plasmid escaped the replication block at 42°C by being inserted into pR325, there was no SOS induction. SOS induction by the arrest of miniF replication required the miniF lynA ⁺ locus in cis, the host recA ⁺ and lexA ⁺ genes. We found that SOS induction was increased greatly near the stationary phase and that cell viability declined. During host cell exponential growth, miniFts9 and miniFts17 plasmids were lost rapidly, although SOS induction persisted for several cell generations. We postulate that lynA expresses a persistent product that may lead to the unwinding of chromosomal DNA.     

Physical mapping and characterization of the mitochondrial DNA and RNA sequences from mit- mutants defective in cytochrome oxidase peptide 1 (OXI 3).

Summary The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA ⁺ lexA ⁺-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication.The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3.Induced stable DNA replication was found to be considerably more resistant to UV irradiation than nromal replication both in a uvrA6 strain and a uvr ⁺ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions

Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli. Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell repiration. On the contrary, both recA⁺ and recA441 cultures either treated with bleomycin or incubated at 42°C failed to induce any of the different SOS functions cited above.Furthermore, after bleomycin addition or thermal treatment both recA⁺ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth. The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied.     

Prophage induction in thermosensitive DNA mutants of Bacillus subtilis

Summary Incubation of thermosensitive dna mutants of Bacillus subtilis at the non-permissive temperature leads in some instances to induction of defective prophage PBSX and cell lysis. A clear distinction can be made between mutants affected in DNA replication at the growing point (extension mutants) and those unable to initiate new rounds of replication (initiation mutants). The former promote PBSX induction to a variable and mutation-specific extent, whereas the latter do not exhibit any signs of induction. Analysis of mutants carrying two dna mutations suggests that products of some dna genes involved in initiation and in extension are not essential for induction but can substantially amplify its extent. However, mitomycin C treatment of dna mutants which have completed their residual DNA synthesis leads to a PBSX induction essentially identical to that obtained by mitomycin C treatment of the wild-type strain, which precludes an essential role for any of the mutated proteins in this induction process. On the basis of our observations we propose that the induction signal is related to the number of blocked replication forks: the larger that number, the higher the proportion of induced cells within the population.     

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae Lz4W

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double‐strand breaks by RecA‐dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold‐sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold‐sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV‐sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction‐like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re‐initiation. This model is consistent with our observation that low temperature‐induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli

Summary To measure the degree of phr gene induction by DNA-damaging agents, the promoter region was fused to the coding region of the lacZ gene in plasmid pMC1403. The new plasmids were introduced into Escherichia coli cells having different repair capabilities. More efficient induction of phr gene expression was detected in a uvrA ^– strain as compared with the wild-type strain. In addition, obvious induction was detected in uvrA ^– cells treated by 4-nitroquinoline 1-oxide and mitomycin C. Nalidixic acid, an inhibitor of DNA gyrase, also induced phr gene expression. In contrast, little induced gene expression was noted in UV-irradiated lexA ^– and recA ^– strains. It is suggested from these results that induction of the phr gene is one of the SOS responses. Possible nucleotide sequences which could be considered to constitute an SOS box were found at the regulator region of the phr gene.Abbreviations phr photoreactivation - UV ultraviolet light - 4NQO 4-nitroquinoline 1-oxide - MMC mitomycin C - PRE photoreactivating enzyme - E. coli Escherichia coli     

Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse

Elevated mistranslation induces a mutator response termed translational stress‐induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyV tRNA gene (Asp→Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA‐dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaV^Glu tRNA (Glu→Ala mistranslation), in which the mutator phenotype is recA‐independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under‐appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.     

Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression.

Summary The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or -rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976). Synthesis of large quantities of protein X is induced by UV, -rays, MMC treatment or tif expression in rec ⁺ but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA ⁺ cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation.Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage or septation.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Summary Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42° C but not at 30° C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA ⁺ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA ⁺. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

The SOS response is induced by replication fork blockage at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites

A new model system for the study of the SOS response has been developed. In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids. Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter. SOS induction can, therefore, be controlled by arabinose. The extent of the SOS response was monitored by measuring the activity of β-galactosidase, expressed from a lacZ gene fused to the 5′ region of the sfiA gene, a typical SOS-responsive gene. Expression of the fusion gene is completely dependent on recA ⁺ and lexA ⁺ genes. Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction. The properties of this system are discussed. Received: 10 May 1998 / Accepted: 20 May 1999     

Induction of recA+-protein synthesis in Escherichia coli.

Summary Escherichia coli was infected with precA ⁺to determine the genetic and physiological factors controlling recA ⁺gene expression. When precA ⁺replication was prevented by superinfection immunity, recA ⁺protein synthesis was induced by UV radiation. The recA ⁺gene is negatively controlled by the lexA ⁺gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA ⁺protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA ⁺protein synthesis. Conversely positive control of recA ⁺gene expression requires recA ⁺protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA ⁺protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA ⁺protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA ⁺protein. lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA ⁺protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA ⁺and recA ⁺proteins normally combine to repress the recA ⁺gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA ⁺protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as repressor.