Sodium arsenite inhibits spontaneous and induced mutations in Escherichia coli

Sodium arsenite at a non-toxic concentration was found to inhibit strongly mutagenesis induced by ultraviolet light (UV), 4-nitroquinoline-1-oxide (4NQO), furylfuramide (AF-2) and methyl methane-sulfonate (MMS) as well as spontaneous mutation in the reversion assay of E. coli WP2uvrA/pKM101. The effect was not, however, seen in the case of the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In order to elucidate the mechanism of the mutation-inhibitory effect of sodium arsenite, its action on umuC gene expression and DNA-repair systems was investigated. It was found that sodium arsenite depressed beta-galactosidase induction, corresponding to the umuC gene expression. For UV-irradiated E. coli strains possessing different DNA-repair capacities, sodium arsenite decreased the UV survival rates of WP2, WP2uvrA[uvrA] and WP67[uvrA polA], increased those of SOS-uninducible strains having either the recA+ or uvrA+ such as CM571 [recA], CM561 [lexA(Ind-)] and CM611[uvrA lexA (Ind-)], and did not affect that of the uvrA recA double mutant, WP100. From these results, we assume that sodium arsenite may have at least two roles in its antimutagenesis: as an inhibitor of umuC gene expression, and as an enhancer of the error-free repairs depending on the uvrA and recA genes.     

Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds.

A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E. coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds. Pretreatment with a sublethal dose (60 microM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde. CAR was also observed in WP2uvrA (uvrA-) and ZA12 (umuC-) cells, but not in ZA60 (recA-) and CM561 (lexA- (Ind-] cells. A role of recA and lexA in CAR was further suggested by the lack of beta-galactosidase induction in recA- and lexA- cells by H2O2. CAR and beta-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA- cells, but no induction of beta-galactosidase by H2O2 was observed in cells with the same gene transfer. These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response.     

Mutagenicity of methyl-, ethyl-, propyl- and butylnitrosourea towards Escherichia coli WP2 strains with varying DNA repair capabilities.

Methyl- (MNUA), ethyl- (ENUA), propyl- (PNUA) and butylnitrosourea (BNUA) have been tested for toxicity and mutation in a liquid suspension assay towards Escherichia coli WP2 and some of its repair deficient derivatives. A comparison of survival rates after nitrosourea exposure between WP2 and WP2 uvrA showed no difference between the two strains but a consistent difference in potency between the various nitrosoureas studied. Toxicity increased in the order MNUA less than PNUA less than ENUA less than BNUA. ENUA and PNUA induced a greater number of trp+ revertants in both strains than did MNUA and BNUA, particularly at low survival rates. None of these differences in biological potency could be accounted for by differences in rates of hydrolysis. ENUA, PNUA and BNUA were non-mutagenic towards WP2 lexA, WP2 recA and WP2 uvrA lexA, whereas MNUA did induce mutations. Ethyl methanesulphonate (EMS) was able to mutate WP2 lexA. These results are discussed in the light of current theories regarding the mechanism of action of these compounds.     

Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities.

Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb [BLB]) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA). With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis. In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated. The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex+ rec+ repair may play a major role in mutagenesis by both BLB and 254-nm radiation. The hypothesis that BLB radiation selectively inhibits rec+ lex+ repair was tested by sequential BLB-254-nm radiation. With strain WP2, a fluence of 30 J/m2 at 254 nm induced trp+ revertants at a frequency of 15 X 10(-6). However, when 10(5) J/m2 or more of BLB radiation preceded the 254-nm exposure, no trp+ revertants could be detected. A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA). However, strains WP2s (uvrA) and wP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given.     

recA-dependent DNA repair processes

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli.

The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic. UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain. The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host. The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain.     

Mutagenic activity of certain synthetic steroids: structural requirement for the mutagenic activity in Salmonella and E. coli

Eight steroids, structurally related to cholesterol, were tested for mutagenic activity in the Ames tester strains. All the test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA and lexA of Escherichia coli was observed as compared to their wild-type counterpart in the presence of the steroids. The role of recA and lexA genes gains further support from the lambda prophage induction in the lysogen as well as with Salmonella strains triggering the error-prone SOS response. Structural features which appear to be essential for mutagenic activity in these strains of the steroids are (1) reactive thio, sulfonyl or sulfinyl groups at the 6 position and (2) a halogen group at the 3 position of the steroidal nucleus. The mutagenicity appears to involve the formation of H2O2 as well as superoxide and hydroxyl radicals.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

Effect of Photoreactivation on the Filling of Gaps in Deoxyribonucleic Acid Synthesized After Exposure of Escherichia coli to Ultraviolet Light

Daughter-strand gaps in deoxyribonucleic acid (DNA) synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled during subsequent incubation in buffer, and the rate of filling is increased when the incubation in buffer is carried out in the presence of 360-nm light. It is concluded that daughter-strand discontinuities are prevented from being rapidly sealed in the dark not because of some structural feature of the daughter-strand but because of the presence of a pyrimidine dimer on the opposite (parental) strand. "Photoreactivation-stimulated gap filling" is dependent on the polA(+) and recA(+) but not the exrA(+) genes. It is suggested that the removal of the dimer allows gap-filling by DNA polymerase I and polynucleotide ligase. The recA(+) gene may be needed at a very early stage, possibly for gap stabilization.     

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

The effect of gaseous nitrogen dioxide (NO2) 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 NO2 (90, 180 microliter/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 microliter/l NO2 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. NO2 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 NO2 mutagenesis in the cells treated with bubbling of NO2 gas. NO2 caused mutation to Trp+ of WP2.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli.

Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.     

Recombinational DNA repair: the ignored repair systems

The recent finding of a role for the recA gene in DNA replication restart does not negate previous data showing the existence of recA-dependent recombinational DNA repair, which occurs when there are two DNA duplexes present, as in the case for recA-dependent excision repair, for postreplication repair (i.e., the repair of DNA daughter-strand gaps), and for the repair of DNA double-strand breaks. Recombinational DNA repair is critical for the survival of damaged cells.     

Lethla effect of nitrous acid on Escherichia coli.

The effect of nitrous acid (NA) on viability, integrity of cellular DNA and on membrane transport were studied in 5 strains of Escherichia coli. Stationary phase cells, grown on mineral salts medium, were exposed to NA. The viability of strains decreased in thefollowing order: W3110 wild-type greater than WP2 wild-type, WP2 uvrA greater than NG30 recA greater than P3478 polA. Alterations were found in the DNA sedimentation profile in alkaline sucrose gradient. Disturbance of DNA synthesis was measured by 3H-labelled thymidine ([3H]Thd) incorporation. No degradation of DNA was found after NA treatment. Low doses of NA caused significant inhibition of leucine and glucose transport into whole cells. The results are interpreted in terms of the multi-target action of NA causing the death of cells.