High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction

Escherichia coli is generally resistant to H(2)O(2), with >75% of cells surviving a 3-min challenge with 2.5 mM H(2)O(2). However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H(2)O(2), with <1% surviving the challenge. Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay. Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H(2)O(2) into a hydroxyl radical, the direct effector of DNA damage. The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold. In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed. This action permits free iron to redox cycle rapidly in the presence of H(2)O(2), thereby augmenting the rate at which hydroxyl radicals are formed. During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage. Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants. More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state.     

Polyphosphate accumulation and oxidative DNA damage in superoxide dismutase-deficient Escherichia coli.

Inorganic polyphosphate is a ubiquitous, linear polymer of phosphate residues linked by high-energy phosphoanhydride bonds. In response to starvation, polyP levels are increased up to 100-fold. It has been proposed that chelation of transition metals by polyP might reduce their toxicity, and that polyP accumulation is vital for survival in stationary phase. SOD-deficient E. coli is unable to survive in stationary phase. We found that deletion of the cytoplasmic SODs does not impair the cell's capability of synthesizing polyP. However, transient accumulation of polyphosphate correlated with increased resistance to H(2)O(2) and protection of DNA against oxidative damage. The reason for this protective effect of polyP is the induction of HPII catalase and DNA repair enzymes as members of the rpoS regulon. PolyP did not directly protect DNA against oxidative damage in vitro and acted as a pro-oxidant by stimulating the production of hydroxyl radical in the Fenton reaction. It is thus suggested that accumulation of poly P and rpoS induction cannot compensate for the lack of cytosolic SODs for survival in stationary phase.     

A mechanism by which nitric oxide accelerates the rate of oxidative DNA damage in Escherichia coli

The presence of nitric oxide (NO) greatly accelerates the rate at which hydrogen peroxide (H2O2) kills Escherichia coli. Workers have suggested that this effect may be important in the process of bacteriocide by phagocytes. The goal of this study was to determine the mechanism of this synergism. The filamentation of the dead cells, and their protection by cell-permeable iron chelators, indicated that NO/H2O2 killed cells by damaging their DNA through the Fenton reaction. Indeed, the number of DNA lesions was far greater when NO was present during H2O2 exposure. In the Fenton reaction, free intracellular iron transfers electrons from adventitious donors to H2O2, producing hydroxyl radicals. Although NO damaged the [Fe-S] clusters of dehydratases, this did not increase the amount of free iron and was therefore not the reason for acceleration of Fenton chemistry. However, NO also blocked respiration, an event that previous studies have shown can stimulate oxidative DNA damage. The resultant accumulation of NADH accelerates the reduction of free flavins by flavin reductase, and these reduced flavins drive Fenton chemistry by transferring electrons to free iron. Indeed, mutants lacking the respiratory quinol oxidases were sensitive to H2O2, and NO did not have any further effect. Further, mutants that lack flavin reductase were resistant to NO/H2O2, and overproducing strains were hypersensitive. We discuss the possibility that H2O2 and NO synergize when macrophages attack captive bacteria.     

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.     

A DNA damage checkpoint in Escherichia coli

An early attempt to find out if the DNA double helix is actively unwound before being replicated was not conclusive, but it did disclose the existence of a unique moment in the life cycle of Escherichia coli when the cell registers whether or not its DNA is intact. If not, the cell embarks on rapid breakdown of its DNA, like "apoptosis" in eukaryotic cells.     

Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli

Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.     

A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII

Prokaryotes and lower eukaryotes possess redundant activities that remove the plethora of oxidative DNA base damages produced during normal oxidative metabolism and which have been associated with cancer and aging. Thus far, only one oxidized pyrimidine-specific DNA glycosylase has been identified in humans, hNthl. Here, we report the identification of three new putative human DNA glycosylases that are phylogenetically members of the Fpg/Nei family primarily found in the bacterial kingdom. We have characterized one of these, hNEI1, and show it to be functionally homologous to bacterial Nei, that is, its principal substrates are oxidized pyrimidines, it undergoes a lyase reaction by, beta,delta-elimination and traps a Schiff base with a substrate containing thymine glycol (Tg). Furthermore, inactivation of active site residues shown to be important in Escherichia coli Nei inactivate the human enzyme. The hNEI1 gene is located on the long arm of chromosome 15 that is frequently deleted in human cancers.     

Body iron is a contributor to oxidative damage of DNA

The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08–0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = ? 0.13 (95% CI ? 0.21 to ? 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects.     

Mechanism of damage recognition by Escherichia coli DNA photolyase

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.     

Body iron is a contributor to oxidative damage of DNA

The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08-0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = - 0.13 (95% CI - 0.21 to - 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects.     

Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis

The effect of visible light on Escherichia coli H10407 in seawater microcosms was investigated. Light damage was estimated by loss of colony-forming ability. Illumination of E. coli suspended in oligotrophic seawater with visible light at an intensity of about 40 klux caused a drastic decrease of culturable bacteria which turned to a viable but non-culturable state. In seawater E. coli exhibited weak metabolic activity as estimated by ³H methyl-thymidine incorporation in the cell. Visible light did not significantly alter this metabolic activity and did not involve detectable oxidation of lipid membranes as evaluated by gas chromatography analysis of fatty acids. The involvement of oxygen and reactive oxygen species in phototoxicity was studied. A decrease of the toxic effect was observed when E. coli was exposed to visible light under anaerobic conditions. Scavengers of reactive oxygen species exhibited variable protective effects. β-Carotene, a singlet oxygen scavenger, and superoxide dismutase were equally ineffective. On the other hand, catalase, which eliminates hydrogen peroxide and thiourea, a hydroxyl radical scavenger, showed a net protection. In addition desferrioxamine B, an iron chelator, was also effective in reducing phototoxicity, probably by preventing hydroxyl radical generation by decomposition of hydrogen peroxide in the presence of iron (Fenton reaction). Therefore, hydrogen peroxide and hydroxyl radical seem to be reactive intermediates of oxygen-dependent (type II) photosensitized reactions.     

Repair of nitrous acid damage to DNA in Escherichia coli.

A number of mutant strains of Escherichia coli have been examined for their sensitivity to nitrous acid and in some instances to methylmethanesulfonate. All ung- mutants tested are abnormally sensitive to nitrous acid. Since the ung mutation is phenotypically expressed as a defect in uracil DNA glycosidase, this observation supports the contention that treatment of cells with nitrous acid causes deamination of cytosine to uracil. In addition the observed sentitivity indicates that the ung gene is involved in the repair of uracil in DNA. Studies with other mutants suggest that both exonuclease III and DNA polymerase I of E. coli are involved in the repair of nitrous acid damage in vivo.     

RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli

The small RNA RyhB has recently been shown to negatively regulate a number of mRNAs encoding dispensable iron-using proteins in Escherichia coli. The resulting decrease in the synthesis of iron-using proteins is thought to spare iron in order to ensure its availability for iron-requiring proteins that are indispensable. Indeed, the expression of RyhB from a heterologous promoter activates the iron-sensing repressor Fur, which suggests an increase in the pool of free intracellular iron (iron-sparing). In accordance with these observations, we report here that RyhB expression increases the concentration of free intracellular iron, as shown by direct measurements of the metal in whole cells by electron paramagnetic resonance spectroscopy. Our data also suggest that iron-sparing originates from rapid uptake of extracellular iron and not from already internalized metal. Furthermore, RyhB is shown to be essential for normal bacterial growth and survival during iron starvation, which is consistent with previous data describing the function of the small RNA. Overall, our data demonstrate that, by regulating synthesis of nonessential iron-using proteins, the small RNA RyhB ensures that the iron is directed towards the iron-requiring enzymes that are indispensable.     

Near-ultraviolet radiation blocks SOS responses to DNA damage in Escherichia coli

Summary Escherichia coli cells in which the recA promoter is fused to a lac structural gene, (Mu) Mud(Ap,lac)::rec, were irradiated with two far-ultraviolet light wavelengths (254 and 290 nm), selected monochromatic near-ultraviolet (NUV) wavelengths 313 nm, 334 nm, 365 nm, or broad band solar-UV (290–420 nm) from a solar simulator. Irradiation with the two far-ultraviolet wavelengths was followed by high yields of -galactosidase, lambda prophage induction, and Weigle reactivation. These end points were not observed after irradiation with the selected NUV wavelengths or the broad spectrum solar-UV. Thus, neither broad spectrum solar-UV nor monochromatic NUV wavelengths resulted in the derepression of the recA promoter. Further, prior exposure of the cells either to the selected monochromatic NUV wavelengths or to solar-UV inhibited (a) the induction of -galactosidase by subsequent 254-nm radiation, (b) subsequent 254-nm induction of lambda prophage, (c) Weigle reactivation, and (d) mutation frequency. These observations are consistent with the hypothesis that NUV blocks subsequent recA protease action. Offprint requests to: Ms. Susan Barr, Editor, Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IL 60439, USA     

The nature of the damage to Escherichia coli DNA induced by gamma-irradiation.

Quantitative studies of the number of gamma-induced single-strand breaks (SSBs) and enzyme-labile sites (ELSs) were performed using the model of Col E1 plasmids, which undergo transition from the covalently closed form (CCF) into the open circular form (OCF) during gamma-irradiation of the plasmid-bearing strain E. coli JC 411. By adding 0.5 MEDTA the repair endonucleases of the cell, which effect the transition of ELSs into SSBs during and after gamma-irradiation, were totally inhibited. It was found thless than 15 per cent of the number of gamma-induced lesions are primarily induced SSBs. About the saditions of direct radiation damage. The conclusions are that (1) the contribution of the direct radiation effect in the cell is greater than that of the indirect effect; (2) the main type of gamma-induced lesions are the ELSs (most of which--more than 75 per cent--are alkali-stable; (3) the enzymatic incision of gamma-induced ELSs into SSBs is effected very quickly, mainly during irradiation; and (4) 0.5 MEDTA is a universal inhibitor of repair processes in cell, including the action of N-glycosidases and endonucleases.