Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1–3 mM) of hydrogen peroxide (H₂O₂) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H₂O₂-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of ΔrecA cells carrying plasmid-borne recA (P_tac-recA⁺) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind⁻) genetic background or inducible in a lexA⁺ background. At a H₂O₂ dose resulting in maximal killing, ΔrecA lexA3 (Ind⁻) cells with P_tac-recA⁺ show 40-fold greater survival than lexA3 (Ind⁻) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and ΔrecA lexA⁺ cells with P_tac-recA⁺. To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H₂O₂-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

Borrelia burgdorferi vlsE antigenic variation is not mediated by RecA

RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.     

The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli

In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.     

recA730-dependent suppression of recombination deficiency in RecA loading mutants of Escherichia coli

Homologous recombination is an essential process in double-strand break repair. The main requirement for recombination is formation of a RecA filament. Double-strand breaks can be processed into a RecA filament by the action of three enzymatic activities: helicase, 5′-3′ exonuclease and RecA loading onto ssDNA. These activities are provided by the RecBCD enzyme in wild type cells or by the RecF pathway gene products in recBC sbcBC(D) cells. In the recBD1080A mutant (recB∗ mutant), the recombination machineries of RecBCD and RecF pathways are interchangeable and include RecB∗CD enzyme (helicase), RecJ (5′-3′ exonuclease) and RecFOR (RecA loading). The mutant RecA730 protein is able to produce a RecA filament without the help of RecFOR mediators, since it more efficiently competes with SSB protein for ssDNA than the normal RecA protein. It was previously shown that the recA730 mutation suppresses UV sensitivity in a uvrA recFOR genetic background. We tested whether the recA730 mutation can suppress recombination and DNA repair deficiency in a recB∗ mutant and its derivatives. We show that the recA730 mutation suppresses recombination deficiency in a recB∗ recFOR background, where the defect is at the level of RecA loading, but not in the recB∗ recJ background where the defect is at the level of nuclease activity.     

The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.     

Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.     

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX_Hs) can interact with the H. seropedicae RecA protein (RecA_Hs) and that RecA_Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX_Hs inhibited 90% of the RecA_Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA_Hs. RecA_Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX_Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX_Hs protein negatively modulates the RecA_Hs activities by protein-protein interactions and also by DNA-protein interactions.     

The E. coli recA gene can restore the defect in mutagenesis of the pso4-1 mutant of S. cerevisiae

The E. coli recA gene was introduced into the pso4-1 mutant of S. crivisiae and transformants were treated with 8-MOP + UVA and 254-nm UV light. The results showed that the recA gene increased the resistance to the toxic effect of 8-MOP + UVA and restored the frequency of reversion of the pso-4-1 mutants after both treatments. The presence of the recA gene stimulated expression of the small subunit of the ribonucleotide reductase (Rnr2) in the pso4-1 mutants. Thus the E. coli recA gene is functional in yeast. Moreover, it was shown that pso4-1 mutant is epistatic to pso-1 and rad6-1, which belong to a mutagenic repair pathway. We propose here that the PSO4 gene has some role in the control of mutagenic repair in yeast.     

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.     

Analysis of recA mutants with altered SOS functions

The Escherichia coli RecA protein has at least three roles in SOS mutagenesis: (1) derepression of the SOS regulon by mediating LexA cleavage; (2) activation of the UmuD mutagenesis protein by mediating its cleavage; and (3) targeting the Umu-like mutagenesis proteins to DNA. Using a combined approach of molecular and physiological assays, it is now possible to determine which of the three defined steps has been altered in any recA mutant. In this study, we have focussed on the ability of six particular recA mutants (recA85, recA430, recA432, recA433, recA435 and recA730) to perform these functions. Phenotypically, recA85 and recA730 were similar in that in lexA⁺ and lexA(Def) backgrounds, they exhibited constitutive coprotease activity towards the UmuD mutagenesis protein. Somewhat surprisingly, in a lexA(Ind⁻) background, UmuD cleavage was damage inducible, suggesting that the repressed level of the RecA¹ protein cannot spontaneously achieve a fully activated state. Although isolated in separate laboratories, the nucleotide sequence of the recA85 and recA730 mutants revealed that they were identical, with both alleles possessing a Glu³⁸ → Lys change in the mutant protein. The recA430, recA433 and recA435 mutants were found to be defective for both λ mutagenesis and UmuD cleavage. λ mutagenesis was fully restored, however, to the recA433 and recA435 strains by a low copy plasmid expressing the mutagenically active UmuD′ protein. In contrast, λ mutagenesis was only partially restored to a recA430 strain by a high copy UmuD′ plasmid, suggesting that RecA430 may also be additionally defective in targeting the Umu proteins to DNA. Sequence analysis of the recA433 and recA435 alleles revealed identical substitutions resulting in Arg²⁴³ → His. The recA432 mutation had a complex phenotype in that its coprotease activity towards UmuD depended upon the lexA background: inducible in lexA⁺ strains, inefficient in lexA(Ind⁻) cells and constitutive in a lexA(Def) background. The recA432 mutant was found to carry a Pro¹¹⁹ → Ser substitution, a residue believed to be at the RecA subunit interface; thus this complex phenotype may result from alterations in the assembly of RecA multimers.     

The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences

The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli. RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair. Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences—several of which exhibit enhanced RecA protein-promoted homologous pairing activity. The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias. Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart. This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein. We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.     

The sir locus of Escherichia coli: a gene involved in SOS-independent repair of mitomycin C-induced DNA damage

A Mud1 (lac Ap^r) insertion has been isolated in a Δ(lac)reaA⁺ lexA3(Ind^-rpoB87 gyrA87 mutant of Escherichia coli resulting in a decrease in mitomycin C tolerance and an increase in post-mitomycin C DNA degradation. The mitomycin C sensitivity of the insertion mutant is not further increased by substituting either the rpoB87 or the gyrA mutation by the respective wild-type alleles. However, when both rpoB87 and gyrA87 mutations are replaced by rpoB⁺ and gyrA⁺ the strain becomes hypersensitive to mitomycin C. Inactivation of recA in the insertion mutant has no effect on its mitomycin C sensitivity provided both rpoB87 and gyrA87 are present. When either or both of the mutations is/are replaced by the wild-type allele inactivation of recA renders the strain hypersensitive to mitomycin C. The locus of Mud1 (lac Ap^r) insertion, designated sir (SOS-independent repair), has been mapped between 57 and 61 min on the E. coli linkage map. Expression of the sir gene seems to be constitutive and not enhanced by mitomycin C. These results are discussed in relation to the SOS-independent repair of mitomycin C-induced DNA damage reported earlier.     

The Escherichia coli recA gene increases UV-induced mitotic gene conversion in Saccharomyces cerevisiae

The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceeding mitotic conversion events in yeast.     

Critical role of RecN in recombinational DNA repair and survival of Helicobacter pylori

Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10- and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.     

Does RecA have a role in Borrelia recurrentis?

Genomic sequencing of two relapsing fever spirochaetes showed truncation of recA in Borrelia recurrentis, but not in Borrelia duttonii. RecA has an important role among bacteria; we investigated whether this characteristic was representative of B. recurrentis, or an artefact following in vitro cultivation. We sequenced recA directly from samples of patient with louse-borne relapsing fever (B. recurrentis) or tick-borne relapsing fever (B. duttonii). We confirmed the premature stop codon in seven louse-borne relapsing fever samples, and its absence from three tick-borne relapsing fever samples. Furthermore, specific signature polymorphisms were found that could differentiate between these highly similar spirochaetes.