Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Induction of intracellular endonuclease synthesis in Serratia marcescens by agents suppressing DNA replication]

Endonuclease synthesis in Serratia marcescens was studied in the presence of agents selectively suppressing DNA biosynthesis: nalidixic acid, mitomycin, hydroxyurea and thymine limitation. All the agents suppressing DNA replication induced exocellular endonuclease biosynthesis irrespective of their action mechanism. The greatest inducing effect was exerted when the agents were added to cells in the late exponential phase. Endonuclease biosynthesis was induced 1-2 hours after adding the agent and was inhibited with chloramphenicol. The induction of exocellular endonuclease synthesis in Serratia marcescens by the classical inducing agents of a SOS response seems to be indicative of a Lex A regulated process.     

Influence of nalidixic acid on killing and mutation of V79 cells exposed to different damaging agents

The effect of treatment with nalidixic acid, an inhibitor of DNA topoisomerase, after exposure of V79 cells to different DNA-damaging agents on the induction of killing and mutation has been studied. The DNA-damaging agents were ultraviolet light, gamma-rays and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). It was seen that treatment with nalidixic acid potentiated the killing by MNNG and suppressed the induction of mutation. However, it had no influence upon killing and mutation by UV light and gamma-rays. The difference in the observed results could be due to the nature of the damage induced and its repair in relation to the function of topoisomerases.     

Enhanced survival of gamma-irradiated Escherichia coli following pretreatment with dithiothreitol

Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT). The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157. The strains were incubated in 10 mmol dm-3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT. Following the expression period the DTT-incubated cells and incubated control cells were irradiated. When AB1157 cells were pretreated with chloramphenicol (200 micrograms cm-3) for a period of 30 min prior to addition of the induction media no increase in survival was seen. When catalase (0.1 mg cm-3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0.893 (ER-1 = 1.12). Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1.7 with DTT present and 1.3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1.0. Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT. Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1.7 with DTT present during irradiation and 1.3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1.0. Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

Mechanistic analysis of the contributions of DNA and protein damage to radiation-induced cell death

Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Alkylating Agents Induce Uvm, a Reca-Independent Inducible Mutagenic Phenomenon in Escherichia Coli

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing δrecA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.     

Involvement of recA and exr Genes in the In Vivo Inhibition of the recBC Nuclease

When Escherichia coli cells are gamma irradiated they degrade their deoxyribonucleic acid (DNA). The DNA of previously gamma-irradiated T4 phage is also degraded in infected cells. The amount of degradation is not only dependent on the dose but also on the genotype of the cell. The amount of degradation is less in cells carrying a recB or a recC mutation, suggesting that most of the DNA degradation is due to the recB(+) and recC(+) gene product (exonuclease V). In some strains a previous dose of ultraviolet (UV) light followed by incubation renders the cells resistant to DNA degradation after gamma irradiation. We have shown this inhibition to take place for infecting T4 phage also. By using six strains of E. coli selected for mutations in the genes recA, exr (or lex), and uvrB, we have been able to show that the preliminary UV treatment produces no change in recA and exr cells for both endogenous DNA degradation and the degradation of infecting irradiated T4 phage DNA, i.e., inhibition was not detected in these strains. On the other hand, wild-type cells and strains carrying mutations of uvrB show inhibition in both types of experiments. Because the recA gene product and the exr(+) (lex(+)) gene product are necessary for the induction of prophage, it is possible that the phenomenon of inducible inhibition requires recA(+) and exr(+) presence. One interpretation of these results is that an inducible inhibitor may be controlled by the exr gene.     

Mechanism of action of nalidixic acid on Escherichia coli. IV. Effects on the stability of cellular constituents

Cook, Thomas M. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), William H. Deitz, and William A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. IV. Effects on the stability of cellular constituents. J. Bacteriol. 91:774-779. 1996.-Treatment of Escherichia coli 15TAU with nalidixic acid resulted in degradation of the nucleic acids of the cells, whereas protein was unaffected. Deoxyribonucleic acid (DNA) degradation appeared to be more extensive than ribonucleic acid degradation during periods of comparable bactericidal action. The onset of DNA degradation was evident prior to a measurable bactericidal effect. However, within the range of 2 to 20%, DNA degradation was accompanied by a decrease in viable cell numbers. Degradation of DNA to acid-soluble material occurred only under conditions permitting the bactericidal action of nalidixic acid. Arrest of the bactericidal action of nalidixic acid by the addition of dinitrophenol or chloramphenicol also inhibited DNA degradation. The acid-soluble products, which were excreted into the medium, have not been characterized completely, but probably were not phosphorylated.     

Characterization of an inducible oxidative stress system in Bacillus subtilis.

Exponentially growing cells of Bacillus subtilis demonstrated inducible protection against killing by hydrogen peroxide when prechallenged with a nonlethal dose of this oxidative agent. Cells deficient in a functional recE+ gene product were as much as 100 times more sensitive to the H2O2 but still exhibited an inducible protective response. Exposure to hydrogen peroxide also induced the recE(+)-dependent DNA damage-inducible (din) genes, the resident prophage, and the product of the recE+ gene itself. Thus hydrogen peroxide is capable of inducing the SOS-like or SOB system of B. subtilis. However, the induction of this DNA repair system by other DNA-damaging agents is not sufficient to activate the protective response to hydrogen peroxide. Therefore, at least one more regulatory network (besides the SOB system) that responds to oxidative stress must exist. Furthermore, the data presented indicate that a functional catalase gene is necessary for this protective response.     

The involvement of topoisomerases and DNA polymerase I in the mechanism of induced thermal and radiation resistance in yeast

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation.     

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.     

Changes in the survival curve shape of E. coli cells following irradiation in the presence of uncouplers of oxidative phosphorylation

Four uncouplers of oxidative phosphorylation (UOP) (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, 4-hydroxybenzylidenemalonitrile and N-phenylanthranilic acid) have been found to alter the shape of the radiation survival curves of several cell lines of E. coli when present during irradiation in oxia. Incubation of cells with high concentrations of UOP for 30 min before irradiation induced an increase in extrapolation number (n) in cell lines AB 1157 (wild-type), AB 1886(uvrA-) and KMBL(polA-) but not GR 501(lig-)ts, AB 2463(recA-) and AB 2480(uvrA-recA-). In addition the UOP all effect a decrease in mean lethal dose (D0) even when tested at low concentrations or short contact times. Studies with wild-type cells correlate the increase in n with measured increased levels of ATP (above oxic control cells) produced upon incubation with UOP. The increased levels of ATP most likely arise from the UOP overstimulating glycolysis. The decrease in D0 cannot be associated with any of the repair pathways investigated and it is concluded that the highly lipophilic UOP directly or indirectly potentiate other target(s) to radiation damage as well as DNA under oxic conditions. Treatment of the cells with UOP did not result in the deleterious depletion of energy substrates, loss of non-protein thiols or the production of cytotoxins upon irradiation.