Induction Of SOS Functions Research Articles

Abstract 1328: Replication inhibition and mutagenicity of 8,5’-cyclopurine-2’-deoxynucleosides in Escherichia coli

Abstract 8,5’-Cyclopurine-2’-deoxynucleosides are unique tandem DNA lesions formed by oxidation or ionizing radiation, which are repaired only by nucleotide excision repair. They have been suspected to be responsible for neurodegeneration and other diseases. But their replication properties are little studied. In the current work, we have evaluated replication of a pMS2 plasmid containing a single (5's)-8,5’-cyclo-2’-deoxyadenosine (5's-cdA) or (5's)-8,5’-cyclo-2’-deoxyguanosine (5's-cdG) at a preselected site in Escherichia coli. In the wild type E. coli cells, viability of 5's-cdA and 5's-cdG plasmids dropped to approximately 1% and 0.5%, respectively, relative to control. The viability of each was further reduced in pol II-deficient strain, but it was most pronounced in a pol V-deficient strain, where it dropped ten-fold. By contrast, viability increased 4-7-fold in a pol IV-deficient strain. Upon induction of SOS functions, viability increased ten-fold in the wild type strain. Though less pronounced, viability increased in the other strains as well. We conclude that pol V is necessary for translesion synthesis of the cyclopurines and that it may work cooperatively with pol II, which is also needed for bypass. In addition, it appears that although pol IV competes with the other bypass polymerases, it is inefficient in bypassing these lesions. In the absence of SOS, 5's-cdA-to-G mutations occurred at a low frequency, whereas 5's-cdG-to-A events were detected at a much higher frequency. With SOS, 5's-cdA-to-T and 5's-cdG-to-T were also detected. [Supported by NIEHS grant ES-013324] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1328. doi:10.1158/1538-7445.AM2011-1328

The induction of SOS function in Escherichia coli K-12/PQ37 by 4-nitroquinoline oxide (4-NQO) and fecapentaenes-12 and -14 is bile salt sensitive: implications for colon carcinogenesis

The response of Escherichia coli to genotoxic agents involves the triggering of a complex system of genes known as the SOS response. In E. coli PQ37, a test organism used for the assessment of genotoxicity, lacZ, the β-galactosidase gene is placed under the control of sfiA, one of the SOS genes through an operon fusion. The induction of β-galactosidase activity, when the organism is exposed to genotoxic agents, is an indirect measure of the genotoxic activity of the test compound. Incubation of E. coli PQ37 with either 4-nitroquinoline oxide (4-NQO) or one of the fecal mutagens, fecapentaene-12 or -14 (F-12 or F-14) in the presence of sodium taurocholate or sodium deoxycholate resulted in a significant enhancement of induction of β-galactosidase activity. The molecular mechanisms of 4-NQO-induced mutagenesis in E. coli are similar to those of the effects of UV light in which both replication-dependent and repair-dependent pathways of mutagenesis exist. Since E. coli PQ37 is excision-repair-deficient, alternate pathways are involved in this system. Bile salts by themselves do not trigger the SOS response, and hence their role in enhancing the SOS-inducing potency of mutagens may involve the potentiation of the cleavage-inactivation of lexA (repressor of SOS) by the protein product of the SOS-controlled gene, recA. The potentiating effect of bile salts on the fecal mutagens, F-12 and F-14, has implications in their suspected role in colon carcinogenesis associated with high-fat, low-fiber diets.

Mutagenic potential of stereoisomeric bay region (+)- and (-)-cis-anti-benzo[a]pyrene diol epoxide-N2-2'-deoxyguanosine adducts in Escherichia coli and simian kidney cells.

We have investigated the mutagenic potential of site-specifically positioned DNA adducts with (+)- and (-)-cis-anti stereochemistry derived from the binding of r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) to N2-2'-deoxyguanosine (G1 or G2) in the sequence context 5'TCCTCCTG1 G2CCTCTC. BPDE-modified oligodeoxynucleotides were ligated to a single-stranded DNA vector and replicated in Escherichia coli or simian kidney (COS7) cells. The presence of (+)- or (-)-cis adduct strongly reduced the yield of transformants in E. coli, and the yield was improved by the induction of SOS functions. Both adducts were mutagenic in E. coli and COS cells, generating primarily G --> T transversions. In E. coli, the (-)-cis adduct was more mutagenic than the (+)-cis adduct, while in COS cells, both adducts were equally mutagenic. These results were compared with those obtained with stereoisomeric (+)- and (-)-trans adducts [Moriya, M., et al. (1996) Biochemistry 35, 16646-16651). In E. coli, cis adducts, especially (-)-cis adducts, are consistently more mutagenic than the comparable trans adduct. In COS cells, trans adducts yield higher frequencies of mutations than the two cis adducts and, with the exception of the high-mutation frequency associated with the (+)-trans adduct at G2, relatively small differences in mutation frequencies are observed for the three other adducts. In E. coli, mutation frequency is a pronounced function of adduct stereochemistry and adduct position. These findings suggest that the fidelity of translesional synthesis across BPDE-dG adducts is strongly influenced by adduct stereochemistry, nucleotide sequence context, and the DNA replication complex.

Fidelity of Translesional Synthesis past Benzo[a]pyrene Diol Epoxide−2‘-Deoxyguanosine DNA Adducts: Marked Effects of Host Cell, Sequence Context, and Chirality

We have used a site-specific approach to investigate the mutagenic potential of (+)- and (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adducts. Oligodeoxyribonucleotides (5'TCCTCCTG1G2-CCTCTC), modified at the exocyclic amino groups of G1 or G2, were incorporated into a single-stranded shuttle vector and introduced into Escherichia coli or simian kidney (COS) cells. This experimental system permits translesional synthesis to proceed in the absence of DNA repair. The presence of (+)- or (-)-BPDE-N2-dG adducts strongly inhibited translesional synthesis in E. coli; induction of cellular SOS functions reduced this blocking effect. Vectors containing (+)-BPDE adducts at G1 or G2 generated mutation frequencies of 19% and 3%, respectively; these values were not altered significantly by induction of SOS functions. In COS cells, (+)-BPDE-modified vectors generated mutation frequencies of 13% at G1 and 45% at G2. In E. coli, the (-)-BPDE adduct generated mutation frequencies of < or = 2% at G1 and G2 and, in COS cells, 13% at G1 and 21% at G2. The predominant mutations in E. coli and COS cells were G-->T transversions targeted to the site of the lesion; however, when G2 was modified, a significant number of targeted G-->A and G-->C mutations were observed in COS cells. We conclude from this study that (+)-and (-)-BPDE-N2-dG adducts pair preferentially to dCMP and dAMP during translesional synthesis in a process that is strongly influenced by the stereochemistry of the adduct, by the bases flanking the lesion, and by host cell factors.

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions

Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.

On the mechanisms of genotoxicity and metabolism of quercetin.

Quercetin has been the subject of numerous studies on its genetic toxicity and carcinogenicity. Despite its well-proven genetic damaging activity for various genetic end-points (reverse mutations, induction of SOS functions, induction of sister chromatid exchanges, chromosomal aberrations and micronuclei), the mechanisms of genetic damage by quercetin remain, by and large, unknown. The present study aims to further extend the observations on the possible active oxygen species mediated DNA-damaging activity of quercetin and the role of cytochrome P450-dependent metabolism on the genotoxicity of quercetin. The results reported in this work show that quercetin can produce the OH. radical, as assessed by deoxyribose degradation in the presence of Fe3+/EDTA (ethylenediaminetetraacetic acid), and that it induces strand breakage in isolated plasmidic DNA (pUC18). The data support the hypothesis that the production of OH. is mediated by H2O2. The results with genetically engineered V79 cells expressing rat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolism of quercetin, as indicated by the fact that neither an enhancement nor a decrease in the genotoxicity of quercetin was observed. Results obtained on the pH dependence of the induction of chromosomal aberrations by quercetin in V79 cells show that, as the pH value of the medium is increased to 8.0, there is a significant increase in the number of aberrant cells, as expected if oxygen radicals are responsible for the formation of chromosomal aberrations.

Regulation in repressor inactivation by RecA protein

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate. We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-gamma-S. The active ternary complex is formed by binding of ATP-gamma-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-gamma-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a phi 80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the phi 80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a phi 80cI-lambda chimeric repressor. These results strongly suggested that the active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.

Characterization and genotoxicity of DNA adducts caused by 2-naphthyl isocyanate

Calf thymus DNA and M13mp9 RF DNA were modified with [ring-3H]2-naphthyl isocyanate (NIC) and analyzed by reverse-phase HPLC following enzymatic hydrolysis. In each case, essentially, a single radioactive component, which co-chromatographed with authentic N4-2-naphthyl-carbamoyl-2'-deoxycytidine (NCdC), was detected. In order to explore the biological potential of this adduct, mp9 RF DNA modified with NIC was introduced into Escherichia coli strains using a calcium chloride technique. The plaque-forming efficiencies of DNA decreased with increasing adduct level, and the decreases were more pronounced in Uvr endonuclease-deficient strains (i.e. AB1886, uvrA; AB1885, uvrB; AB1884, uvrC) as compared to JM103 (Uvr endonuclease proficient) and JM101 RH03 (recA). These results suggest that these lesions, NCdC adducts, can be removed by the Uvr endonuclease repair system. Mutations were detected as the loss of ability of the bacteriophage to complement the defective beta-galactosidase of the host cells. Induction of SOS functions in the host cells enhanced the mutation frequency to 0.089%, i.e. greater than or equal to 4-fold greater than in non-SOS-induced cells, in transfections with RF DNA that contained 100 adducts/molecule. The mutagenic potency of this cytidine lesion is lower than that of the guanine-C8 adducts of 2-aminofluorene and 2-acetylaminofluorene as reported previously for this mutagenesis system.

Anti-mutagenic effect of ultraviolet light on spontaneous tyrosine tRNA ochre suppressor mutations in Escherichia coli.

The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered. The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form. We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations. With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation. As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein). The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized.

Induction of SOS function in Escherichia coli by some mycotoxins

AbstractThe genotoxic activity of 11 mycotoxins was investigated in Escherichia coli K12. The induction of the SOS function s fi A whose level of expression is monitored by means of a s fi A :: lac Z operon fusion was assayed by measuring the β‐galactosidase activity in the PQ 37 strain. Most of these fungal metabolites did not induce SOS response in this bacterial test. Only aflatoxicol, a reduced metabolite of aflatoxin B1, was well detected as an SOS inducer if metabolic activation was performed. Patulin, penicillic acid, and viomellein were only weak inducing agents. The other fungal compounds tested failed to demonstrate a positive SOS inducing activity. The relationship between the SOS Chromotest, mutagenicity to Salmonella typhimurium, and in vivo carcinogenicity was discussed.

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.

Induction of SOS Functions in Escherichia coli and Biosynthesis of Nitrosamine in Rabbits by Nitrogen Dioxide

Induction of SOS Functions in Escherichia coli and Biosynthesis of Nitrosamine in Rabbits by Nitrogen Dioxide

Induction of SOS functions in Escherichia coli and biosynthesis of nitrosamine in rabbits by nitrogen dioxide.

Nitrogen dioxide induced SOS functions in Salmonella typhimurium and Escherichia coli K-12 and was mutagenic in Escherichia coli WP2. When a rabbit was administered aminopyrine intravenously and administered nitrogen dioxide by inhalation, N-nitrosodimethylamine was detected in its blood. Analysis was conducted with 15N-nitrosodimethylamine as an internal standard by a combination of capillary gas chromatography and mass spectrometry. Accompanying administration of cystamine increased the blood concentration of N-nitrosodimethylamine in the rabbit, suggesting inhibition of its metabolism. Concurrent sulfur trioxide inhalation increased N-nitrosodimethylamine formation in the rabbit.

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities ( uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway ( uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.

Bacterial DNA reactivity of the novel antitumor antibiotics PD 114,759 and PD 115,028 (veractamycins A and B).

The novel antitumor antibiotics PD 114,759 and PD 115,028 were evaluated for their ability to cause repairable DNA damage and the induction of SOS functions in bacterial systems. PD 114,759 and PD 115,028 were preferentially toxic to DNA repair-defective Escherichia coli WP100 uvrA recA in comparison to wild-type E. coli WP2 at concentrations of 10 approximately 30 micrograms/ml in agar diffusion assays. Both compounds were inducers of cell filamentation and prophage lambda (two E. coli SOS functions) at concentrations of 0.1 approximately 1 microgram/ml. In addition, the ability of PD 114,759 and PD 115,028 to retain their filamentation-inducing effects under both aerobic conditions and anaerobic conditions suggests that a bioreductive, rather than an oxygen-requiring, mechanism is involved in the DNA-reactive effects of these agents.

Induction of SOS functions by alkaline intracellular pH in Escherichia coli.

Alkalinization of intracellular pH (pHi) causes an increase in UV resistance in wild-type and pH-sensitive mutant (DZ3) cells of Escherichia coli. Utilizing cells transformed with a plasmid (pA7) which bears the uvrA promoter fused to galK galactokinase structural gene, it was shown that alkaline pHi leads to an increase in the specific activity of galactokinase. This effect was not displayed in a mutant bearing a recA-insensitive lexA gene, nor in cells harboring a plasmid (pA8) in which the galK is fused to a lexA-insensitive uvrA promoter. Hence, the effects of pHi on cells functions may involve the lexA product of the SOS system.

Induction of SOS functions by nitrogen dioxide in Escherichia coli with different DNA-repair capacities

The effect of gaseous nitrogen dioxide (NO 2) on cytotoxicity, induction of synthesis of UmuC and RecA proteins, and mutagenesis was studied in Escherichia coli strains with different capacities of DNA repair. Gaseous NO 2 (90, 180 μl/l) killed Escherichia coli. The recA mutant was most sensitive, the lexA mutant moderately sensitive, and the uvrA mutant and the wild-type the least sensitive. When 90 μl/l NO 2 gas was bubbled into bacterial suspensions for 30 min at a flow rate of 100 ml/min, the induction of umuC gene expression increased in the wild-type strain. NO 2 also induced the recA gene expression in the wild-type strain. The synthesis of neither RecA nor UmuC proteins was induced in the recA and lexA mutants. We further investigated the NO 2 mutagenesis in the cells treated with bubbling of NO 2 gas. NO 2 caused mutation to Trp + of WP2.

Role of the uvr gene in the SOS function inducing activity of two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, in Escherichia coli K12

Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS-chromotest using PQ 37 ( uvr A) and PQ 35 ( uvr +) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 < Trp-P-2 < aflatoxin B1, at low concentrations (<125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr + strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp-P-1 and Trp-P-2. At higher concentrations (125–500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration.

Induction of SOS functions is not recquired for recA+-dependent mutagenicity of 9-aminoacridine in Salmonella typhimurium strain trpE8

Induction of SOS functions is not recquired for recA+-dependent mutagenicity of 9-aminoacridine in Salmonella typhimurium strain trpE8

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage λ and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the λc1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70–90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.