The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa

Inactivation of the Pseudomonas aeruginosa mutM, mutY , or mutT gene conferred a 2.4-, 17.2-, or 38.1-fold increase in spontaneous mutation frequency, respectively. Importantly, the mutY and mutT strains each displayed a robust H₂O₂-induced mutation frequency. In addition, the mutM, mutY , and mutT mutations severely sensitized P. aeruginosa to killing by H₂O₂, suggesting that these gene products act to repair one or more cytotoxic lesions in P. aeruginosa . Nucleotide sequence analysis of a fragment of the rpoB gene from rifampicin resistant mutM -, mutY -, and, mutT -deficient strains was consistent with this conclusion. These findings are discussed in terms of possible roles for mutM, mutY , and mutT in contributing to survival and mutagenesis of P. aeruginosa colonizing the airways of cystic fibrosis patients.     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Differential sensitivities of transformed and untransformed murine cell lines to DNA cross-linking agents relative to repair of O6-methylguanine

Sensitivities of several murine cell strains to killing by the DNA cross-linking agents 1-(2-chloroethyl)-1-nitrosourea (CNU), cis-diamminedichloroplatinum (II) (Cis-Pt) and mitomycin C (MMC) were measured by post-treatment colony-formation. Virally-transformed murine cells were usually more sensitive to cell killing by these agents than were the parental 3T3 cell strains. The hypersensitivity to CNU of some virally-transformed murine cell strains correlated well with the reduced ability to repair O6-methylguanine (O6mGua), a phenomenon similar to that in human cells. The loss of ability to repair O6mGua, as well as the increased sensitivity of transformed strains to cell killing, may not be due to a mutation but rather due to a change of gene expression associated with transformation by viruses or activation of oncogenes.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

Lethal and mutagenic action of hydrogen peroxide on Haemophilus influenzae.

The lethal and mutagenic effects of H2O2 on wild-type Haemophilus influenzae Rd and on uvr1, uvr2, rec1, and rec2 mutant strains were studied. The first two mutants are sensitive to UV, and the second two are defective in recombination. Rd, urv1, and rec1 strains were more sensitive to the killing effect of H2O2 treatment than were uvr2 and rec2 strains. There were peaks of mutagenesis at two H2O2 concentrations over a range of 30 to 275 mM. Our results suggest a specific repair of H2O2 damage that is independent of the Uvr2 and Rec2 gene products. Sensitivity to the killing effect of H2O2 and to the lethal action of near-UV light were similar for Rd and uvr1 strains. This finding suggests that the mechanisms of killing by and repair of H2O2 damage may have some overlap with those of near-UV radiation.     

Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

We partially depleted the O6-methylguanine-DNA methyltransferase activity in four O6-methylguanine (O6-mGua) repair-proficient (Mer+) human cell strains with exogenous O6-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O6-mGua from DNA at about half the rate of control cells, while removal of 3-methyladenine was unaffected. In spite of partial depletion of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer+ strains to killing by MNNG, we treated cells with O6-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O6-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer- A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O6-mGua post-treatment was begun, greater than 50% of O6-mGua was already removed from cell DNA. Our findings may be accounted for by at least two schemes, one in which nonlethal O6-mGua are removed from DNA rapidly, while potentially lethal O6-mGua are repaired later. The other scheme proposes that exogenous O6-mGua increases the lethality of a non-O6-mGua lesion by reducing its repair both in Mer+ and Mer- cells. Both schemes are consistent with the hypothesis that O6-mGua may be a lethal DNA lesion in human cells.     

Interactions among the Escherichia coli mutT,mutM, and mutY damage prevention pathways

We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis. mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases. To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles. We report three major findings. First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds. We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A. Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP. Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.     

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.     

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.     

DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

Electronic excited molecular oxygen (singlet oxygen, ¹O₂) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by ¹O₂ in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a ¹O₂-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with ¹O₂-induced DNA damage are linked to the context of the damaged sequence.     

Analysis of spontaneous base substitutions generated in mutator strains of Bacillus subtilis

In the current studies, we investigated base substitutions in the Bacillus subtilis mutT, mutM, and mutY DNA error-prevention system. In the wild type strain, spontaneous mutations were mainly transitions, either G:C --> A:T or A:T --> G:C. Although both transitions and transversions were observed in mutY and mutM mutants, mutM/mutY double mutants contain strictly G:C --> T:A transversions. In the mutT strain, A:T --> C:G transversion was not observed, and over-expression of the B. subtilis mutT gene had no effect on the mutation rate in the Escherichia coli mutT strain. Using 8-oxo-dGTP-induced mutagenesis, transitions especially A:T --> G:C were predominant in the wild type and mutY strains. In contrary, transversion was high on mutY and double mutant (mutM mutY). Finally, the opuBC and yitG genes were identified from the B. subtilis chromosome as mutator genes that prevented the transition base substitutions.     

Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli

We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%). The distributions of frequently mutated sites of G:C-->T:A and A:T-->C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.     

Singlet oxygen ((1)Delta(g)O(2)) as the principal oxidant in myeloperoxidase-mediated bacterial killing in neutrophil phagosome.

Intraphagosomal viability of wild type E. coli and lycopene (a powerful (1)O(2) quencher)-producing transformant E. coli was investigated using human polymorphonuclear leukocytes as the cells for phagocytosis of opsonized viable bacteria. While the viability of both wild type and the transformant E. coli decreased very rapidly in the phagosome, but the viability of the lycopene-transformant in phagosomes was about 1.7 times higher than that of wild type E. coli after 5 min of incubation. The results were very similar to the results obtained when E. coli strains were exposed to (1)O(2) generated in myeloperoxidase-H(2)O(2)-Br(-) system (a pure (1)O(2) generating system) at pH 4.5. The reason for HOCl, which may be generated in the myeloperoxidase-H(2)O(2)-Cl(-) system under physiological conditions but does not become involved in bactericidal action, could be explained by the near neutral pH in phagosomes at which bacterial killing by chlorination is extensively attenuated. This is the first report which proved (1)O(2)-mediated bacterial killing in neutrophil-bacterial phagosomal system.     

Hydroxylation of deoxyguanosine at 5' site of GG and GGG sequences in double-stranded DNA induced by carbamoyl radicals

Free radicals generated by chemicals can cause sequence-specific DNA damage and play important roles in mutagenesis and carcinogenesis. Carbamoyl group (CONH 2 ) and its derived groups (CONR 2 ) occur as natural products and synthetic chemical compounds. We have investigated the DNA damage by carbamoyl radicals · (CONH 2 ), one of carbon-centered radicals. Electron spin resonance (ESR) spectroscopic study has demonstrated that carbamoyl radicals were generated from formamide by treatment with H 2 O 2 plus Cu(II), and from azodicarbonamide by treatment with Cu(II). We have investigated sequence specificity of DNA damage induced by carbamoyl radicals using 32 P-labeled DNA fragments obtained from the human c-Ha- ras -1 and p 53 genes. Treatment of double-stranded DNA with carbamoyl radicals induced an alteration of guanine residues, and subsequent treatment with piperidine or Fpg protein led to chain cleavages at 5'-G of GG and GGG sequences. Carbamoyl radicals enhanced Cu(II)/H 2 O 2 -mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in double-stranded DNA more efficiently than that in single-stranded DNA. These results shows that carbamoyl radicals specifically induce hydroxylation of deoxyguanosine at 5' site of GG and GGG sequences in double-stranded DNA.     

Methionine reduces spontaneous and alkylation-induced mutagenesis in Saccharomyces cerevisiae cells deficient in O6-methylguanine-DNA methyltransferase

The exposure of DNA to reactive intracellular metabolites is thought to be a major cause of spontaneous mutagenesis. DNA alkylation is implicated in the above process by the fact that bacterial and yeast cells lacking DNA alkylation-specific repair genes exhibit elevated spontaneous mutation rates. The origin of the intracellular alkylating molecules is not clear; however, S-adenosylmethionine (SAM) has been proposed as one source because it has a reactive methyl group known to methylate proteins and DNA. We supplemented yeast cultures with excess methionine and examined the effects of increased endogenous SAM concentration on spontaneous and alkylation-induced mutagenesis in the absence of various DNA repair pathways. Our results show that either the excess methionine, or the increased SAM produced as a result of this treatment, is able to protect yeast cells from mutagenesis, and that this effect is alkylation-damage-specific. The protective effect was observed only in the mgt1 mutant deficient in the O(6)-methylguanine-DNA repair methyltransferase, but not in the wild type or other DNA repair-deficient strains, indicating that the protection is specific for O-methyl lesions. Thus, our results may lend support to the recently reported chemopreventive effect of SAM in rodents and further suggest that the observed tumor prevention by SAM may be, in part, due to its suppression of spontaneous mutagenesis in mammals. Given that a strong correlation has been established between O(6)-methylguanine and carcinogenicity, this study may offer a novel approach to preventing carcinogenesis.     

Recovery of mitomycin C-treated mouse 10T1/2 cells during confluent holding

The relationship between cytotoxicity, sister-chromatid exchanges (SCE) and the repair of DNA crosslinks was studied in mouse 10T1/2 cells during confluent holding following either acute or protracted MMC treatment. No cytotoxic effects were observed with increasing doses of MMC until SCE frequencies 1.8 times background levels were induced. Protracted MMC treatments were less cytotoxic than acute MMC exposure at doses which yielded similar frequencies of SCE. The kinetics of recovery during confluent holding in acute MMC-treated cells were similar for cytotoxicity and the repair of DNA interstrand crosslinks. These results suggest that a type of non-lethal DNA damage which causes SCE may persist for long periods of time in MMC-treated cells. This non-lethal damage may accumulate during protracted MMC exposure while damage leading to cell killing is repaired.     

Dose-dependent regulation of superoxide anion on the proliferation, differentiation, apoptosis and necrosis of human hepatoma cells: the role of intracellular Ca 2+

Dose-dependent regulation of cellular processes is one important characteristic of signaling molecules. Although recent studies suggest that reactive oxygen species (ROS) may act as in vivo signaling molecules, the dose-dependent regulation of ROS on cellular processes together in one system needs to be evaluated. After treating cells with gradually increased O(2)(-), generated by the hypoxanthine-xanthine oxidase system, it was found that: (i) the proliferation of hepatoma cells firstly increased at 1-2 microM O(2)(-), then decreased markedly as the concentration increased; (2) at 8 or 16 microM O(2)(-), re-differentiation of hepatoma cells was induced, as indicated by the indices relating to cell malignancy or differentiation, such as cell surface charge, alpha-fetoprotein, gamma-glutamyltranspeptidase, tyrosine-alpha-ketoglutarate transaminase, cAMP, and the tumor's clonogenic potential; (iii) at 16 microM O(2)(-), accompanied by the re-differentiation of cells, cell apoptosis was also simultaneously induced as indicated by the appearance of apoptotic bodies, detached cells, and other apoptotic morphological features, as well as specific DNA fragmentation; (iv) at the highest concentration of O(2)(-) (32 microM) in this study, cell necrosis was dramatically induced as shown by Trypan blue exclusion; (v), an increase of intracellular Ca(2+) ([Ca(2+)](i)) was observed at all concentrations of O(2)(-) treatment, and this [Ca(2+)](i) increase was found to be involved in the regulation of O(2)(-) on the cellular processes. In conclusion, these results indicate that O(2)(-) could dose-dependently regulate the processes of cells, where Ca(2+) is one of its molecular targets, and hence provide a direct support for the hypothesis that ROS themselves are important signaling molecules.     

Epistatic roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in coping with reactive oxygen species-induced DNA damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.     

Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.