uvrA Gene Research Articles

A consensus sequence for the Rhodospirillaceae SOS operators

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN 7GTAC and GAACN 7GAAC motifs present upstream of the recA gene and the GTTCN 7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.

UV light induces IS10 transposition in Escherichia coli.

A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10. Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies. UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition. Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition. UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome. UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and DeltarecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition. IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions. To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria.

The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria

Deinococcus radiodurans ( Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC S) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation ( mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS 4 of Escherichia coli ( Ec) and to a resolvase found in IS Xc5 of Xanthomonas campestris ( Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS 2621.

Expression of the Pseudomonas aeruginosa uvrA gene is constitutive

By complementation of an Escherichia coli uvrA-defective mutant, the uvrA gene of Pseudomonas aeruginosa has been isolated from a genomic library. The nucleotide sequence of the promoter and the 5′-coding region of this gene have been determined. A P. aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, was not present in the 629-bp upstream region of the cloned uvrA gene. Analysis of transcriptional fusions with the lacZ gene demonstrates that, contrary to what happens with its homologous gene of E. coli, the P. aeruginosa uvrA gene is not DNA damage-inducible. However, the UvrA protein must be functional in P. aeruginosa cells because an uvrA-defective mutant is extremely sensitive to UV radiation.

Cloning, nucleotide sequence and transcriptional analysis of the uvrA gene from Neisseria gonorrhoeae.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrA mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrA proteins of other bacterial species. A second open reading frame (ORF259) was identified upstream from, and in the opposite orientation to the gonococcal uvrA gene. Transcriptional fusions between portions of the gonococcal uvrA upstream region and a reporter gene were used to localise promoter activity in both E. coli and N. gonorrhoeae. The transcriptional starting points of uvrA and ORF259 were mapped in E. coli by primer extension analysis, and corresponding sigma70 promoters were identified. The arrangement of the uvrA-ORF259 intergenic region is similar to that of the gonococcal recA-aroD intergenic region. Both contain inverted copies of the 10 bp neisserial DNA uptake sequence situated between divergently transcribed genes. However, there is no evidence that either the uptake sequence or the proximity of the promoters influences expression of these genes.

Cloning, sequencing and expression of the uvrA gene from an extremely thermophilic bacterium, Thermus thermophilus HB8

One of the most important DNA repair systems is the nucleotide (nt) excision repair system. The uvrA gene, which plays an essential role in the prokaryotic excision repair system, was cloned from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) HB8, and its nt sequence was determined. In the amino acid (aa) sequence of Tt UvrA, a characteristic duplicated structure, two nt-binding consensus sequences (Walker's A-type motif) and two zinc finger DNA-binding motifs were found. The aa sequence showed 73% homology with that of Escherichia coli (Ec). These features suggest that Tt has the same excision repair system as Ec. Upon comparison of the Tt and Ec UvrA, some characteristic aa substitutions were found. The numbers of Arg and Pro residues were increased (from 66 to 81 and from 47 to 55, respectively), and the numbers of Asn and Met residues were decreased (from 33 to 18 and from 18 to 11, respectively) in Tt. The Tt uvrA gene was expressed in Ec under control of the lac promoter. Purified UvrA was stable up to 80°C (at neutral pH) and at pH 2–11 (at 25°C)

Isolation and characterization of the Haemophilus influenzae uvrA gene

The uvrA gene of Haemophilus influenzae (Hi) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed 81% identity and 90% similarity with the Escherichia coli UvrA protein. Consistent with a role of Hi uvrA in DNA repair, a Hi uvrA mutant exhibited increased sensitivity of UV irradiation. Furthermore, Hi uvrA was able to complement a mutation in the E. coli uvrA locus.

Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.

Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon.

The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp.

The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens

The single-stranded-DNA-binding (SSB) proteins from Proteus mirabilis and Serratia marcescens were purified from overproducing Escherichia coli strains, which were devoid of their own ssb gene. The strains harboured an endA insertion mutation and a xonA mutation resulting in the absence of endonuclease I and exonuclease I activities from the preparations. The amino acid sequences of the SSB of all three species are nearly identical in the N-terminal parts of the proteins that contain the DNA-binding domain, but differ in the C-terminal parts. Both proteins have an apparent binding-site size of 65 and 35 nucleotides at high and low salt concentrations, respectively. The association-rate constant for binding to poly(dT) is 3.2 x 10(8) M-1 s-1 for P. mirabilis SSB (PmiSSB) and 3.4 x 10(8) M-1 s-1 for S. marcescens SSB (SmaSSB). These binding parameters are very similar to those of E. coli SSB (EcoSSB). The structural similarity of the proteins is also documented by the finding that they can exchange subunits among each other to form mixed tetramers. The transcriptional regulation of the ssb and uvrA genes from P. mirabilis and S. marcescens in SOS-induced E. coli cells was studied using lacZ fusions. While the uvrA genes were inducible, there was no induction of the ssb genes transcribed divergently from the uvrA genes. Apparently, regions with nucleotide sequence similarity to the E. coli SOS-box preceding the ssb genes of P. mirabilis and S. marcescens had no gross effect on the transcription. Studies on growth of the cells and recovery from ultraviolet damage indicate that the heterologous SSB proteins support DNA replication and recombinational DNA repair of E. coli with the same efficiency as the E. coli SSB protein. Interactions with other E. coli proteins involved in these processes either do not occur, or are not impeded.

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 J m-2, which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6-4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5 Mrad), D. radiodurans can mend > 100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.

Heat-shock-increased survival to far-UV radiation in Escherichia coli is wavelength dependent

Heat-shock-induced resistance to far-UV (FUV) radiation was studied in Escherichia coli. The induction of FUV resistance was shown to be dependent on the products of the genes uvrA and polA in bacteria irradiated at 254 nm. Heat shock increased the resistance to 280 nm radiation in a uvrA6 recA13 mutant. Heat shock lowered the mutation frequency (reversion to tryptophan proficiency) in wild-type or uvrA strains irradiated at 254 nm. When these strains were irradiated at 280 nm, heat shock did not interfere with the mutation frequency in the wild-type strain, but greatly enhanced mutations in the uvrA mutant. After heat-shock treatment, the wild-type strain irradiated at 254 nm showed increased DNA degradation, indicating enhanced repair activity. However, heat shock did not stimulate SOS repair triggered by FUV. An increased survival of bacteriophages irradiated with FUV and inoculated into heat-shock-treated bacteria was not detected. The possibility that heat shock enhances excision repair activity in a wavelength-dependent manner is discussed.

Isolation of Brucella abortus ssb and uvrA genes from a genomic library by use of lymphocytes as probes

Brucella abortus proteins from virulent S2308 expressed from a pBluescript II SK- genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total of 300 clones were evaluated. Ten clones were identified that stimulated T-lymphocyte proliferation. Among them, one clone with a 2.5-kb insert stimulated T-lymphocyte proliferation in all three animals, suggesting that the proteins encoded by genes within this fragment may represent immunodominant antigens. DNA sequencing of this clone reveals two large open reading frames (ORFs). ORF II has a high degree of similarity to the Escherichia coli ssb gene, which codes for the single-stranded DNA binding protein. ORF I, in the opposite direction to ORF II, shows similarity to the N terminus of the E. coli uvrA gene, which codes for one of the three subunits of the E. coli ABC excision nuclease. The observation that the PBM cells recognized and proliferated in response to proteins expressed from single clones provides a novel strategy to select bacterial antigens that may prove useful in designing alternative vaccines against brucellosis.

Enhancement of the uvrA gene dosage reduces pyrimidine dimer excision in UV-irradiated Escherichia coli

E. coli possesses an efficient repair mechanism able to remove pyrimidine dimers from UV-irradiated DNA, which is catalyzed by UvrABC endonuclease. In E. coli B/r Hcr + cells transformed with a multicopy plasmid harboring a gene coding for UvrA, the excision capacity was greatly reduced. The course of thymine dimer excision was investigated using the enzymatic as well as the radiochromatographic method and the results are discussed in term of nonspecific interaction between the excess of UvrA protein and undamaged DNA duplex.

Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA.

The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins. It also has two zinc finger motif regions and two ATP binding sites. To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis. Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E. coli strain deleted of the uvrA gene. One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain. To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif. The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations. Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I. However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA. Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected. Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif. Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes.

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.

Inducible stable DNA replication in Escherichia coli uvr − and uvr − cells treated with genotoxic chemicals

Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E. coli PQ35 and its uvrA derivative E. coli PQ37. In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells. This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it.

Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon.

The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative stress regulon in response to superoxide-generating (redox-cycling) agents. We have cloned the soxR locus, which is positioned near the uvrA gene at 92.2 min on the genetic map, by monitoring complementation of a delta soxR mutation. Subclones from the soxR region in the delta soxR strain simultaneously restored cellular resistance to the redox-cycling agent phenazine methosulfate and inducibility of at least two of the regulon proteins, glucose-6-phosphate dehydrogenase and endonuclease IV, by paraquat, another redox-cycling agent. DNA sequence analysis revealed the presence of two genes involved in activating the soxR regulon. These genes, named soxR and soxS, are arranged divergently with their 5' ends separated by only 85 bp. The predicted 12.9-kDa SoxS protein is related to the AraC family of one-component gene regulators, but corresponds only to the putative DNA-binding regions of these proteins. The 17.1-kDa SoxR protein bears significant homology only to the MerR family of proteins including a predicted DNA-binding helix-turn-helix and a cluster of cysteine residues positioned similarly to those that regulate the activity of MerR in response to Hg2+. This suggests that SoxR could be a metal-binding gene regulator that acts as the intracellular sensor for superoxide. SoxS is evidently the proximal activator of the regulon genes: antibiotic resistance and high-level expression of at least three of the regulon proteins was effected in vivo by the individual expression of SoxS, but not of SoxR, whether or not the cells were exposed to paraquat. These data, together with the recently reported paraquat-inducibility of the soxS gene (Wu, I., and Weiss, B. (1990) J. Bacteriol. 173, 2864-2871), indicate that SoxR and SoxS may constitute a novel type of two-component regulatory system in which the two proteins act sequentially to activate transcription of the various regulon genes in response to superoxide stress.

The single-stranded DNA-binding protein of Escherichia coli

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.

The single-stranded DNA-binding protein of Escherichia coli.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.