recA Gene Product Research Articles

Expression and Characterization of the Staphylococcus aureus RecA protein: A mapping of canonical functions

Recombinases are responsible for homologous recombination (HR), proper genome maintenance, and accurate deoxyribonucleic acid (DNA) duplication. Moreover, HR plays a determining role in DNA transaction processes such as DNA replication, repair, recombination, and transcription. Staphylococcus aureus, an opportunistic pathogen, usually causes respiratory infections such as sinusitis, skin infections, and food poisoning. To date, the role of the RecA gene product in S. aureus remains obscure. In this study, we attempted to map the functional properties of the RecA protein. S. aureus expresses the recA gene product in vivo upon exposure to the DNA-damaging agents, ultraviolet radiation, and methyl methanesulfonate. The recombinant purified S. aureus RecA protein displayed strong single-stranded DNA affinity compared to feeble binding to double-stranded DNA. Interestingly, the RecA protein is capable of invasion and formed displacement loops and readily performed strand-exchange activities with an oligonucleotide-based substrate. Notably, the S. aureus RecA protein hydrolyzed the DNA-dependent adenosine triphosphate and cleaved LexA, showing the conserved function of coprotease. This study provides the functional characterization of the S. aureus RecA protein and sheds light on the canonical processes of homologous recombination, which are conserved in the gram-positive foodborne pathogen S. aureus.

Visualization of UV-induced Replication Intermediates in <em>E. coli</em> using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in <em>E. coli</em> using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria.

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.

Environmental regulation of recA gene expression in Porphyromonas gingivalis

The recA gene product in Porphyromonas gingivalis is involved in DNA repair. Further, disruption of this gene can affect the proteolytic activity and expression of other virulence factors in this organism. Since several known environmental factors can influence virulence gene expression in P. gingivalis, we investigated the influence of these signals on the expression of the recA gene in this organism. A heterodiploid strain of P. gingivalis (designated FLL118) containing a transcriptional fusion of the recA promoter region and the promoterless tetracycline-resistant gene [tetA(Q)2] and xylosidase/arabinosidase (xa) gene cassette was constructed. The recA promoter activity was assessed by measurement of xylosidase activity in FLL118. The expression remained relatively constant during different growth phases, at different pH levels and in the presence of DNA-damaging agents. In response to hemin limitation and in the presence of calcium there was a moderate increase in recA promoter activity. Temperature also affected the expression. The highest level of xylosidase activity was observed in cultures at 32 degrees C with a decline of approximately 46% as growth temperature increased to 41 degrees C. Reverse transcriptase polymerase chain reaction analysis revealed that this regulation may be occurring at the transcriptional level. These results suggest that expression of the recA gene in P. gingivalis W83 is responsive to several environmental signals but is not regulated by a DNA damage-inducible SOS-like regulatory system.

Mutagenesis and more: umuDC and the Escherichia coli SOS response.

The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and post-translational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

Protein Partners of the BRCA1 Tumor Suppressor

The primary amino acid sequence of BRCA1 offers few clues about the mechanism by which it suppresses tumor formation in normal breast and ovarian tissues. In an effort to unravel its biological functions, investigators have sought to identify the proteins that interact with BRCA1 in vivo. These efforts have already uncovered two interacting proteins: the BRCA1-associated RING domain (BARD1) protein, a novel polypeptide that bears a striking structural resemblance to BRCA1, and hRAD51, a human homolog of the bacterial recA gene product. As proliferating cells enter S phase of the cell cycle, the BRCA1, hRAD51, and BARD1 polypeptides aggregate in discrete nuclear domains, commonly described as "BRCA1 nuclear dots''. However, when S phase cells sustain DNA damage, the dots are mobilized such that BRCA1 and its associated proteins relocate to sites of replicating DNA. Thus, as a participant in the cellular response to DNA damage, BRCA1 may suppress tumor formation by preserving the integrity of genomic DNA.

Molecular characterization ofGluconobacter oxydans recAgene and its inhibitory effect on the function of the host wild-typerecAgene

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.

Molecular characterization of Gluconobacter oxydans recA gene and its inhibitory effect on the function of the host wild-type recA gene

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.

DNA damage-dependent recruitment of nucleotide excision repair and transcription proteins to Escherichia coli inner membranes.

The entire process of nucleotide excision repair (NER) in Escherichia coli has been reconstituted in vitro from purified proteins and defined DNA substrates. However, how this system is organized in vivo in unclear. We report here the isolation and characterization of macromolecular assemblies containing NER and transcription proteins from E. coli. This ensemble consists of at least 17 proteins. They are recruited, as a consequence of DNA damage induced by UV irradiation, to the inner membrane. The UV-induced 6-4 photoproducts are also relocated to the inner membrane following UV-irradiation of the cells. This recruitment process is dependent on the uvrA, uvrC and recA gene products. These results suggest that at least part of the repair process may associate with the inner membrane and also provide insights into understanding the cellular organization of repair processes.

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.

Direct Association between the Yeast Rad51 and Rad54 Recombination Proteins

The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH2-terminal 115 residues of the Rad54 protein are necessary for this interaction. Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.

Thiols, rec A induction and radiosensitivity in Escherichia coli

Induction by gamma-radiation, UV radiation or hydroxyurea of RecA gene product synthesis in Escherichia coli, monitored as beta-D-galactosidase in recA-lacZ fusion strains, was shown to be inhibited if 2-mercaptoethylamine (MEA) was added before treatment with the inducing agents. If cysteine (Cys) at low concentrations was added at the same time as MEA it counteracted the action of MEA. The effect of MEA may be described as a competitive inhibition of an inducing or conducting effect of Cys. In E. coli GE499 (uvrA+), complete inhibition by 30-mmol dm-3 MEA of recA induction was associated with about five times higher radio-resistence. Both of these effects of MEA were completely reversed by 0.3-mmol dm-3 Cys. As shown in parallel experiments with E. coli GE500 (uvrA-), these effects of MEA and Cys were shown to be independent of excision-repair proficiency. Treatment of bacteria with MEA and/or Cys was shown not to lead to increased intracellular concentrations of these thiols. Instead, treatment with them appeared to provoke conspicuous increases in glutathione levels, which are, however, probably not directly involved in the studied action of MEA and Cys.

β subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli

Exposure of Escherichia coli to UV irradiation of nalidixic acid, which induce both the SOS and heat shock responses, led to a 3–4-fold increase in the amount of the β subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-β antibodies. Such an induction was observed also in a Δ rpoH mutant lacking the heat shock-specific σ 32 subunit of RNA polymerase, but it was not observed in aecA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the β subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the β subunit, to the lacZ reporter gene showed induction of β-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5′ flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the β subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.

Selection or mutation: Which, if either, comes first?

Selection-induced mutations (SIMS) are mutations that occur as specific and direct responses to environmental challenges, and that occur more often when they are selectively advantageous than when they are selectively neutral. This review includes discussions of how the occurrence of SIMS is measured, acquisitive evolution by SIMS, some of the controversies surrounding SIMS and models that have been advanced to explain the specificity of SIMS, and the requirement of a functional recA gene product for SIMS to occur.

Efficiency of homologous intermolecular recombination at different locations on the Bacillus subtilis chromosome

The efficiencies of intermolecular recombination at 12 different locations on the Bacillus subtilis chromosome were determined by transforming competent cells with a nonreplicative plasmid. The efficiencies varied by only about threefold but were significantly different (P less than 0.05 by a chi-square test) for approximately 20% of the locations. The recA gene product is required for recombination, and the addA gene product appears to affect the variation in a site-specific way.

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway.

Incubation of UV-irradiated plasmid DNA with a protein extract prepared from Escherichia coli cells led to the production of mutations in the cro gene residing on the plasmid. The mutations were detected in a subsequent bioassay step, which involved transformation of an indicator strain with the plasmid DNA that was retrieved from the reaction mixture, followed by plating on lactose/MacConkey plates. UV mutations produced in this cell-free reaction required the recA and umuC gene products and were prevented by rifampicin, an inhibitor of RNA polymerase, which inhibited plasmid replication. Removal of pyrimidine photodimers from the plasmid by enzymatic photoreactivation after the in vitro stage, but prior to transformation, increased plasmid survival as expected. Surprisingly, it also caused a large increase in the frequency of UV mutations detected in the bioassay. This photoreactivation-stimulated in vitro UV mutagenesis was dependent on the excision repair genes uvrA, uvrB, and uvrC and occurred in the absence of DNA replication. This suggests that two distinct UV mutagenesis pathways occurred in vitro: a replication-dependent pathway (type I) and a repair-dependent pathway (type II). DNA sequence analysis of type II UV mutations revealed a spectrum similar to that of in vivo UV mutagenesis. When the photoreactivation step was included in the protocol, type II UV mutagenesis did not require the RecA and UmuC proteins. These results are in agreement with the in vivo delayed photoreactivation phenomenon, where the removal of photodimers after an incubation period eliminated the requirement for RecA and UmuC in UV mutagenesis. The above system will enable the biochemical analysis of UV mutagenesis and the isolation of proteins involved in the process.

Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene

The effects of the expression of Escherichia coli truncated RecA protein on the host recA functions were examined. The recA gene on a multicopy plasmid was manipulated to express the truncated RecA protein from its carboxyl (C) and amino (N) terminal ends where a maximum of four extra amino acid residues was added. The regulatory part of the recA gene was substituted by the lacUV5 promoter in the plasmid to facilitate the artificial control of recA expression. Enzyme-linked immunosorbent assay and Western blot analyses revealed great differences in accumulation of the truncated RecA proteins in the cell, depending on the location of the site of truncation. The expression of truncated proteins lacking 62, 77, 93 or 149 amino acid residues from the C-terminal end caused the host recA + wild-type cell to become sensitive to ultraviolet irradiation and interfered with chromosomal recombination but did not interfere with the induction of λ prophage. The expression of truncated RecA protein with 25 amino acid residues deleted from the C-terminal end caused the host cell to induce SOS functions constitutively. Truncated RecA proteins with 15 or 28 amino acid residues missing from the N-terminal end severely interfered with all of the host recA functions examined here. The effect of the loss of 41 amino acid residues from the N-terminal end of RecA was significant but less than the effect of proteins lacking 15 or 28 amino acid residues from the N-terminal end. A protein lacking 59 amino acid residues from the N-terminal end showed little interference with any measured recA functions, suggesting that the deletion of the region from around residues 41 to 59, which is rich in hydrophobic side-chains, influenced the ability of the truncated protein to interfere with the functions of wild-type RecA protein. We also constructed a mutant gene with an internal deletion whose product was missing a region from residues 184 to 204. That mutant RecA protein was stably accumulated in the cell. This protein had little effect on the function of host wild-type recA gene product. The possible function of the regions at the N and C termini are discussed.

Mapping ethanol-induced deletions

Chromosomal rearrangements, uniformly represented by very large deletions, were stimulated upon transiently exposing Escherichia coli cells with a defective lambda prophage to about 18% (v/v) ethanol. It was shown that the ethanol treatment induced deletion formation rather than enriching for ethanol-tolerant cells. The deletions in 435 mutants were mapped to 26 groups. Ethanol treatment changed the spectrum of deletions relative to those arising spontaneously, and stimulated the formation of deletions with endpoints in E. coli DNA flanking the lambda fragment. The promotion of deletion formation by ethanol involves the joining of distant, nonhomologous linear DNA segments, which can be considered an illegitimate recombination event; however, activity of the E. coli recA gene product was also required. Although spontaneous deletions arose in comparable cells defective for recA, the incidence of deletion formation in recA cells was not altered by ethanol. It is proposed that ethanol stimulates chromosomal rearrangements involving two oppositely oriented replication forks, since the localized deletions commonly removed or inactivated the chromosomal segment including the bidirectional lambda origin of replication. The results imply a novel mutagenic process induced by an agent that does not act directly on DNA.

Interaction of RecBCD enzyme with DNA damaged by gamma radiation.

The DNA of a gene 2 mutant (T4 2-) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD+ cells are therefore incapable of supporting the growth of phage T4 2-. Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2(-)-infected bacteria able to form plaques. We found that recBCD+ cells can form plaques if, before infection with T4 2-, they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2- DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of ReCBCD enzyme on T4 2- DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.