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Papers by Bruce Demple

Biochemistry, Jan 26, 2012

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting protein... more DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In the present case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo-form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to about two base pairs. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

BMC Molecular Biology, 2007

Background: Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most... more Background: Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most sensitive indicators of cellular stress. Previous work in human fibroblasts has shown that HO-1 expression is induced by NO, and that transcriptional induction is only partially responsible; instead, the HO-1 mRNA half-life is substantially increased in response to NO. The mechanism of this stabilization remains unknown. In NIH3T3 murine fibroblasts, NO exposure increased the half-life of the HO-1 transcript from ~1.6 h to 11 h, while treatments with CdCl 2 , NaAsO 2 or H 2 O 2 increased the half-life only up to 5 h. Although poly(A) tail shortening can be rate-limiting in mRNA degradation, the HO-1 mRNA deadenylation rate in NO-treated cells was ~65% of that in untreated controls. In untreated cells, HO-1 poly(A) removal proceeded until 30-50 nt remained, followed by rapid mRNA decay. In NO-treated cells, HO-1 deadenylation stopped with the mRNA retaining poly(A) tails 30-50 nt long. We hypothesize that NO treatment stops poly(A) tail shortening at the critical 30-to 50-nt length. This is not a general mechanism for the post-transcriptional regulation of HO-1 mRNA. Methyl methane sulfonate also stabilized HO-1 mRNA, but that was associated with an 8-fold decrease in the deadenylation rate compared to that of untreated cells. Another HO-1 inducer, CdCl 2 , caused a strong increase in the mRNA level without affecting the HO-1 mRNA half-life. The regulation of HO-1 mRNA levels in response to cellular stress can be induced by transcriptional and different post-transcriptional events that act independently, and vary depending on the stress inducer. While NO appears to stabilize HO-1 mRNA by preventing the final steps of deadenylation, methyl methane sulfonate achieves stabilization through the regulation of earlier stages of deadenylation.

Mutagenesis, Nov 9, 2009

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocki... more Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3D and rev7D) or enhanced by (rev1D) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression.

Journal of Bacteriology, Nov 15, 2003

Genomic studies with bacteria have identified redox-responsive genes without known roles in count... more Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage. Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli. Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS. Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins. Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS. Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site. Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription. A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain. Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain. These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

Molecular Cell, Jul 1, 2006

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features ... more Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3 0 -to-5 0 exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3 0 end to reduce the possibility of repair by rejoining the nicked ends.

Journal of Bacteriology, Oct 1, 1997

The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous o... more The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous oxidative damage associated with active aerobic growth. Here we have studied the transcriptional regulation of oxyR in E. coli growing aerobically in rich medium. Expression of a single-copy oxyR::lacZ reporter construct varied sixfold along the growth curve, with the highest value at 4 to 6 h of growth (ϳ14 ؋ 10 8 cells ⅐ ml ؊1 ). Direct measurements of oxyR mRNA by primer extension showed the same biphasic expression but with a peak somewhat earlier in cell growth (2 to 3 h; ϳ3.5 ؋ 10 8 cells ⅐ ml ؊1 ). The results of immunoblotting experiments demonstrated that the level of OxyR protein exhibits the same biphasic expression. Mutant strains lacking adenylate cyclase (cya) or Crp protein (crp) failed to increase oxyR expression during exponential growth. On the other hand, an rpoS mutation allowed oxyR expression to continue increasing as the cells entered stationary phase. Consistent with a biological role for increased levels of OxyR during exponential growth, the crp cya strain had lower activities of catalase hydroperoxidase I and glutathione reductase and an increased sensitivity to exogenously added hydrogen peroxide. These results suggest that the Crp-dependent upregulation of oxyR in exponential phase is a component of a multistep strategy to counteract endogenous oxidative stress in actively growing E. coli cells.

Journal of Bacteriology, Apr 1, 1994

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity ... more Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased 1-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because 0-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase- deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken

Nucleic Acids Research, 1991

The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative... more 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 AsoxR mutation. Subclones from the soxR region in the AsoxR 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 redoxcycling 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.

Journal of Bacteriology, Jun 15, 1999

Escherichia coli by OxyR in response to the metabolic production of H 2 O 2 . Here, we show that ... more Escherichia coli by OxyR in response to the metabolic production of H 2 O 2 . Here, we show that the untranslated oxyS RNA controlled by OxyR has a role in this regulation. The oxyS transcript appears to affect the metabolic output of H 2 O 2 rather than the removal of H 2 O 2 by catalases-hydroperoxidases.

Journal of Bacteriology, Jul 1, 2000

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcript... more The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

Antimicrobial Agents and Chemotherapy, 2001

The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >... more The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >15 genes of this system provide resistance to both oxidants and multiple antibiotics. An association between clinical quinolone resistance and elevated expression of the soxRS regulon has been observed in Escherichia coli, but this association has not been explored for other enteropathogenic bacteria. Here we describe a soxRS-constitutive mutation in a clinical strain of Salmonella enterica (serovar Typhimurium) that arose with the development of resistance to quinolones during treatment. The elevated quinolone resistance in this strain derived from a point mutation in the soxR gene and could be suppressed in trans by multicopy wild-type soxRS. Multiple-antibiotic resistance was also transferred to a laboratory strain of S. enterica by introducing the cloned mutant soxR gene from the clinical strain. The results show that constitutive expression of soxRS can contribute to antibiotic resistance in clinically relevant S. enterica. Antibiotic resistance is an increasing problem in clinical treatment of infectious disease . The acquisition of resistance has been linked to plasmid-borne genes that specify resistance to individual antibiotics (9), chromosomal mutations that alter the cellular targets (33), and activation of bacterial gene expression that confers resistance to diverse agents . Two groups of coregulated genes (regulons) have been associated with chromosomally based resistance to multiple antibiotics in Escherichia coli and Salmonella enterica serovar Typhimurium: the marRAB and soxRS regulons E. A. Martins, P. J. Pomposiello, and B. Demple, unpublished data). In the soxRS system, SoxR protein is activated by oxidation (18) or nitrosylation (11) to trigger transcription of the soxS gene. In the marRAB system, MarR-mediated repression is relieved in response to environmental agents to activate synthesis of MarA (1, 25), a close homolog of SoxS. The SoxS and MarA proteins are the direct activators of genes for resistance to oxidants and antibiotics . Recent studies indicate that SoxS controls more than 15 genes in S. enterica and 39 genes in E. coli (31; P. J. Pomposiello, M. H. J. Bennick, and B. Demple, unpublished data) and that MarA in E. coli controls as many as 60 genes (4). There is some overlap between the soxRS and marRAB regulons, but most of the newly discovered genes are regulated uniquely by one or the other system (4; Pomposiello et al., unpublished data).

Journal of Bacteriology, Feb 1, 1993

The soxRS regulon is a cornerstone of the adaptive defense systems of Escherichia coli against ox... more The soxRS regulon is a cornerstone of the adaptive defense systems of Escherichia coli against oxidative stress. Unexpectedly, activation of this regulon also enhances bacterial resistance to multiple antibiotics that seem unrelated to oxygen radicals. We previously correlated this multiple antibiotic resistance with a reduced rate of synthesis of the OmpF outer membrane porin that does not affect the OmpC or OmpA porins. Studies presented here, with operon and gene fusions of ompF to lacZ, show that the soxRS-dependent repression of

Nucleic Acids Research, 1991

Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focu... more Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis. Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells. The two activities were purified to differing degrees from HeLa cells. One enzyme had the properties of the known HeLa AP endonuclease (Mr -38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein. The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity. This second enzyme may have been detected in other studies but never characterized. In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities. It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected. The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused.

Proceedings of the National Academy of Sciences of the United States of America, Dec 15, 1991

Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucle... more Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles ofthese enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that ludes two bacterial AP endonucleases (ExoA protein of Streptococcus Abbreviations: AP, abasic; MMS, methyl methanesulfonate; IPTG, isopropyl P-D-thiogalactoside. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M80261).

Molecular and Cellular Biology, Sep 1, 1991

The APNI gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease an... more The APNI gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apnl protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apnl (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl-and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apnl mutant yeast cells. Hydrogen peroxide-treated APNI+ but not apnl mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apnl-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apnl. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apnl mutant cells. The rate of spontaneous mutation in apnl mutant S. cerevisiae was 6-to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apnl is substantial and that such lesions can be mutagenic when left unrepaired.

Proceedings of the National Academy of Sciences of the United States of America, Jul 8, 1997

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases ... more Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase ␤ (Pol␤) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Pol␤ and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Pol␤ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Pol␤ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5-terminal deoxyribose 5-phosphate by Pol␤. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

Nucleic Acids Research, 1996

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA d... more Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3′-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IVspecific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo -) and endonuclease IV-overproducing (p-nfo; ∼10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo -strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo -cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo -strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was w5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxypentos-4-ulose residues, but other possibilities are discussed.

Nucleic Acids Research, 1989

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolald... more Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease IlIl and endonu- clease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++- dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000- 52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'- phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'- PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.

Proceedings of the National Academy of Sciences of the United States of America, Oct 1, 1986

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive t... more Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria

Journal of Bacteriology, 2000

Salmonella enterica serovar Typhimurium responds to superoxide-generating agents through soxR-med... more Salmonella enterica serovar Typhimurium responds to superoxide-generating agents through soxR-mediated activation of the soxS gene, whose product, SoxS, is necessary for resistance to oxidative stress. The S. enterica serovar Typhimurium soxRS system also mediates redox-inducible resistance to diverse antibiotics, which may be relevant to clinical infections. In order to identify SoxS-regulated genes in S. enterica serovar Typhimurium, a lacI-regulated expression system for the S. enterica serovar Typhimurium soxS gene was developed. This system was used to demonstrate that soxS expression is sufficient for the induction of resistance to the superoxide-generating drug paraquat and for the transcriptional activation of the sodA and micF genes. In addition, a library of random lacZ insertions was generated and screened for clones displaying differential ␤-galactosidase activity in the presence or absence of SoxS. This selection yielded six independent chromosomal lacZ transcriptional fusions that were activated by either artificial expression of SoxS or exposure of wild-type cells to micromolar concentrations of paraquat. Moreover, disruption of the inducible genes by the insertions rendered S. enterica serovar Typhimurium hypersensitive to millimolar concentrations of paraquat. Nucleotide sequence determination identified the disrupted genes as sodA (Mn-containing superoxide dismutase), fpr (NADPH:ferredoxin oxidoreductase), and ydbK (a putative Fe-S-containing reductase).

Biochemistry, Jan 26, 2012

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting protein... more DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In the present case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo-form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to about two base pairs. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

BMC Molecular Biology, 2007

Background: Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most... more Background: Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most sensitive indicators of cellular stress. Previous work in human fibroblasts has shown that HO-1 expression is induced by NO, and that transcriptional induction is only partially responsible; instead, the HO-1 mRNA half-life is substantially increased in response to NO. The mechanism of this stabilization remains unknown. In NIH3T3 murine fibroblasts, NO exposure increased the half-life of the HO-1 transcript from ~1.6 h to 11 h, while treatments with CdCl 2 , NaAsO 2 or H 2 O 2 increased the half-life only up to 5 h. Although poly(A) tail shortening can be rate-limiting in mRNA degradation, the HO-1 mRNA deadenylation rate in NO-treated cells was ~65% of that in untreated controls. In untreated cells, HO-1 poly(A) removal proceeded until 30-50 nt remained, followed by rapid mRNA decay. In NO-treated cells, HO-1 deadenylation stopped with the mRNA retaining poly(A) tails 30-50 nt long. We hypothesize that NO treatment stops poly(A) tail shortening at the critical 30-to 50-nt length. This is not a general mechanism for the post-transcriptional regulation of HO-1 mRNA. Methyl methane sulfonate also stabilized HO-1 mRNA, but that was associated with an 8-fold decrease in the deadenylation rate compared to that of untreated cells. Another HO-1 inducer, CdCl 2 , caused a strong increase in the mRNA level without affecting the HO-1 mRNA half-life. The regulation of HO-1 mRNA levels in response to cellular stress can be induced by transcriptional and different post-transcriptional events that act independently, and vary depending on the stress inducer. While NO appears to stabilize HO-1 mRNA by preventing the final steps of deadenylation, methyl methane sulfonate achieves stabilization through the regulation of earlier stages of deadenylation.

Mutagenesis, Nov 9, 2009

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocki... more Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3D and rev7D) or enhanced by (rev1D) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression.

Journal of Bacteriology, Nov 15, 2003

Genomic studies with bacteria have identified redox-responsive genes without known roles in count... more Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage. Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli. Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS. Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins. Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS. Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site. Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription. A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain. Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain. These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

Molecular Cell, Jul 1, 2006

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features ... more Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3 0 -to-5 0 exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3 0 end to reduce the possibility of repair by rejoining the nicked ends.

Journal of Bacteriology, Oct 1, 1997

The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous o... more The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous oxidative damage associated with active aerobic growth. Here we have studied the transcriptional regulation of oxyR in E. coli growing aerobically in rich medium. Expression of a single-copy oxyR::lacZ reporter construct varied sixfold along the growth curve, with the highest value at 4 to 6 h of growth (ϳ14 ؋ 10 8 cells ⅐ ml ؊1 ). Direct measurements of oxyR mRNA by primer extension showed the same biphasic expression but with a peak somewhat earlier in cell growth (2 to 3 h; ϳ3.5 ؋ 10 8 cells ⅐ ml ؊1 ). The results of immunoblotting experiments demonstrated that the level of OxyR protein exhibits the same biphasic expression. Mutant strains lacking adenylate cyclase (cya) or Crp protein (crp) failed to increase oxyR expression during exponential growth. On the other hand, an rpoS mutation allowed oxyR expression to continue increasing as the cells entered stationary phase. Consistent with a biological role for increased levels of OxyR during exponential growth, the crp cya strain had lower activities of catalase hydroperoxidase I and glutathione reductase and an increased sensitivity to exogenously added hydrogen peroxide. These results suggest that the Crp-dependent upregulation of oxyR in exponential phase is a component of a multistep strategy to counteract endogenous oxidative stress in actively growing E. coli cells.

Journal of Bacteriology, Apr 1, 1994

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity ... more Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased 1-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because 0-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase- deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken

Nucleic Acids Research, 1991

The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative... more 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 AsoxR mutation. Subclones from the soxR region in the AsoxR 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 redoxcycling 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.

Journal of Bacteriology, Jun 15, 1999

Escherichia coli by OxyR in response to the metabolic production of H 2 O 2 . Here, we show that ... more Escherichia coli by OxyR in response to the metabolic production of H 2 O 2 . Here, we show that the untranslated oxyS RNA controlled by OxyR has a role in this regulation. The oxyS transcript appears to affect the metabolic output of H 2 O 2 rather than the removal of H 2 O 2 by catalases-hydroperoxidases.

Journal of Bacteriology, Jul 1, 2000

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcript... more The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

Antimicrobial Agents and Chemotherapy, 2001

The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >... more The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >15 genes of this system provide resistance to both oxidants and multiple antibiotics. An association between clinical quinolone resistance and elevated expression of the soxRS regulon has been observed in Escherichia coli, but this association has not been explored for other enteropathogenic bacteria. Here we describe a soxRS-constitutive mutation in a clinical strain of Salmonella enterica (serovar Typhimurium) that arose with the development of resistance to quinolones during treatment. The elevated quinolone resistance in this strain derived from a point mutation in the soxR gene and could be suppressed in trans by multicopy wild-type soxRS. Multiple-antibiotic resistance was also transferred to a laboratory strain of S. enterica by introducing the cloned mutant soxR gene from the clinical strain. The results show that constitutive expression of soxRS can contribute to antibiotic resistance in clinically relevant S. enterica. Antibiotic resistance is an increasing problem in clinical treatment of infectious disease . The acquisition of resistance has been linked to plasmid-borne genes that specify resistance to individual antibiotics (9), chromosomal mutations that alter the cellular targets (33), and activation of bacterial gene expression that confers resistance to diverse agents . Two groups of coregulated genes (regulons) have been associated with chromosomally based resistance to multiple antibiotics in Escherichia coli and Salmonella enterica serovar Typhimurium: the marRAB and soxRS regulons E. A. Martins, P. J. Pomposiello, and B. Demple, unpublished data). In the soxRS system, SoxR protein is activated by oxidation (18) or nitrosylation (11) to trigger transcription of the soxS gene. In the marRAB system, MarR-mediated repression is relieved in response to environmental agents to activate synthesis of MarA (1, 25), a close homolog of SoxS. The SoxS and MarA proteins are the direct activators of genes for resistance to oxidants and antibiotics . Recent studies indicate that SoxS controls more than 15 genes in S. enterica and 39 genes in E. coli (31; P. J. Pomposiello, M. H. J. Bennick, and B. Demple, unpublished data) and that MarA in E. coli controls as many as 60 genes (4). There is some overlap between the soxRS and marRAB regulons, but most of the newly discovered genes are regulated uniquely by one or the other system (4; Pomposiello et al., unpublished data).

Journal of Bacteriology, Feb 1, 1993

The soxRS regulon is a cornerstone of the adaptive defense systems of Escherichia coli against ox... more The soxRS regulon is a cornerstone of the adaptive defense systems of Escherichia coli against oxidative stress. Unexpectedly, activation of this regulon also enhances bacterial resistance to multiple antibiotics that seem unrelated to oxygen radicals. We previously correlated this multiple antibiotic resistance with a reduced rate of synthesis of the OmpF outer membrane porin that does not affect the OmpC or OmpA porins. Studies presented here, with operon and gene fusions of ompF to lacZ, show that the soxRS-dependent repression of

Nucleic Acids Research, 1991

Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focu... more Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis. Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells. The two activities were purified to differing degrees from HeLa cells. One enzyme had the properties of the known HeLa AP endonuclease (Mr -38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein. The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity. This second enzyme may have been detected in other studies but never characterized. In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities. It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected. The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused.

Proceedings of the National Academy of Sciences of the United States of America, Dec 15, 1991

Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucle... more Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles ofthese enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that ludes two bacterial AP endonucleases (ExoA protein of Streptococcus Abbreviations: AP, abasic; MMS, methyl methanesulfonate; IPTG, isopropyl P-D-thiogalactoside. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M80261).

Molecular and Cellular Biology, Sep 1, 1991

The APNI gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease an... more The APNI gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apnl protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apnl (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl-and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apnl mutant yeast cells. Hydrogen peroxide-treated APNI+ but not apnl mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apnl-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apnl. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apnl mutant cells. The rate of spontaneous mutation in apnl mutant S. cerevisiae was 6-to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apnl is substantial and that such lesions can be mutagenic when left unrepaired.

Proceedings of the National Academy of Sciences of the United States of America, Jul 8, 1997

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases ... more Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase ␤ (Pol␤) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Pol␤ and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Pol␤ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Pol␤ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5-terminal deoxyribose 5-phosphate by Pol␤. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

Nucleic Acids Research, 1996

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA d... more Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3′-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IVspecific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo -) and endonuclease IV-overproducing (p-nfo; ∼10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo -strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo -cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo -strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was w5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxypentos-4-ulose residues, but other possibilities are discussed.

Nucleic Acids Research, 1989

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolald... more Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease IlIl and endonu- clease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++- dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000- 52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'- phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'- PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.

Proceedings of the National Academy of Sciences of the United States of America, Oct 1, 1986

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive t... more Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria

Journal of Bacteriology, 2000

Salmonella enterica serovar Typhimurium responds to superoxide-generating agents through soxR-med... more Salmonella enterica serovar Typhimurium responds to superoxide-generating agents through soxR-mediated activation of the soxS gene, whose product, SoxS, is necessary for resistance to oxidative stress. The S. enterica serovar Typhimurium soxRS system also mediates redox-inducible resistance to diverse antibiotics, which may be relevant to clinical infections. In order to identify SoxS-regulated genes in S. enterica serovar Typhimurium, a lacI-regulated expression system for the S. enterica serovar Typhimurium soxS gene was developed. This system was used to demonstrate that soxS expression is sufficient for the induction of resistance to the superoxide-generating drug paraquat and for the transcriptional activation of the sodA and micF genes. In addition, a library of random lacZ insertions was generated and screened for clones displaying differential ␤-galactosidase activity in the presence or absence of SoxS. This selection yielded six independent chromosomal lacZ transcriptional fusions that were activated by either artificial expression of SoxS or exposure of wild-type cells to micromolar concentrations of paraquat. Moreover, disruption of the inducible genes by the insertions rendered S. enterica serovar Typhimurium hypersensitive to millimolar concentrations of paraquat. Nucleotide sequence determination identified the disrupted genes as sodA (Mn-containing superoxide dismutase), fpr (NADPH:ferredoxin oxidoreductase), and ydbK (a putative Fe-S-containing reductase).