recA730 Strains Research Articles

Suppression of the E. coli SOS response by dNTP pool changes.

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30+-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

SOS mutator activity: unequal mutagenesis on leading and lagging strands.

A major pathway of mutagenesis in Escherichia coli is mediated by the inducible SOS response. Current models of SOS mutagenesis invoke the interaction of RecA and UmuD'(2)C proteins with a stalled DNA replication complex at sites of DNA lesions or poorly extendable terminal mismatches, resulting in an (error-prone) continuation of DNA synthesis. The precise mechanisms of SOS-mediated lesion bypass or mismatch extension are not known. Here, we have studied mutagenesis on the E. coli chromosome in recA730 strains. In recA730 strains, the SOS system is expressed constitutively, resulting in a spontaneous mutator effect (SOS mutator) because of reduced replication fidelity. We investigated whether during SOS mutator activity replication fidelity might be altered differentially in the leading and lagging strand of replication. Pairs of recA730 strains were constructed differing in the orientation of the lac operon relative to the origin of replication. The strains were also mismatch-repair defective (mutL) to facilitate scoring of replication errors. Within each pair, a given lac sequence is replicated by the leading-strand machinery in one orientation and by the lagging-strand machinery in the other orientation. Measurements of defined lac mutant frequencies in such pairs revealed large differences between the two orientations. Furthermore, in all cases, the frequency bias was the opposite of that seen in normal cells. We suggest that, for the lacZ target used in this study, SOS mutator activity operates with very different efficiency in the two strands. Specifically, the lagging strand of replication appears most susceptible to the SOS mutator effect.

The isfA mutation specifically inhibits the SOS-dependent mutagenic pathway and does not selectively affect any particular base substitution.

We have previously described a new mutation in Escherichia coli, isfA, which causes inhibition of SOS mutagenesis (UV-induced in rec+ and spontaneous in recA730 strains) and several SOS-dependent phenomena. Antimutagenic activity of the isfA mutation in the recA730 strain was shown to be related to inhibition of processing of UmuD to UmuD' by RecA* coprotease. In the present study we have analysed the specificity of the antimutagenic activity of the isfA mutation by employing F' plasmids carrying a set of mutant lacZ genes that can individually detect two types of transitions and four types of transversions. Analysis revealed that isfA inhibits UV-induced G:C-->A:T and A:T-->G:C transitions, but does not affect the same G:C-->A:T transitions induced by EMS, an SOS-independent mutagen. Analysis of the antimutagenic activity of the isfA mutation in two mutator strains, recA730 and mutL, showed that isfA inhibits SOS-dependent transversions in recA730, but not transitions generated as replication errors in the mutL strain. In the double mutant recA730 mutL, both transitions and transversions were enhanced and isfA inhibits most transversions and only those transitions generated by the recA730 mutation. The results indicate that the antimutagenic activity of the isfA mutation is specific for the SOS, UmuD'C-dependent mutagenic pathway but does not selectively affect any particular base substitution. Moreover, studies on the effect of the isfA mutation on transitions and transversions in different genetic backgrounds enable us to recognize different mutagenic pathways active in recA730 cells.

Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains ( mutH, mutS, mutL and uvrD) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of β-galactosidase. In TestD, the strains carrying different recA alleles ( recA +, rcA730 and Δ recA) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac + recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage ( Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpernoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Test B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA + and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.

Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity.

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.

P VII.22 - P VII.22 isfA Mutation inhibits UV-induced GC → AT and AT → GC transitions in recA+ strains and GC → TA and AT → TA transversions in recA730 strains

P VII.22 - P VII.22 isfA Mutation inhibits UV-induced GC → AT and AT → GC transitions in recA+ strains and GC → TA and AT → TA transversions in recA730 strains

The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains.

Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described. We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. The isfA mutation has now been demonstrated also to suppress mutator activity in E. coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.

The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.

Overproduction of the epsilon subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli.

It has been found that the mutator phenotype of the recA441 and recA730 strains that express the SOS response constitutively is suppressed by pIP1, a high-copy plasmid carrying the dnaQ gene encoding the 3'----5' exonuclease subunit (epsilon) of DNA polymerase III. We have constructed plasmid pIP11, in which the dnaQ gene is fused to the strong tac (trp-lac) promoter. Enhanced synthesis of the epsilon subunit stimulated by isopropyl beta-D-thiogalactopyranoside, the inducer of tac, prevents expression of the mutator phenotype of recA441 and markedly decreases the frequency of UV-induced mutations. These results strongly suggest that a loss of editing capacity by the epsilon subunit of DNA polymerase III holoenzyme plays a crucial role in generation of mutations during the SOS response.