Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

The distinct role of catalase and DNA repair systems in protection against hydrogen peroxide in Escherichia coli

The katEkatG mutant of E. coli, UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7. The mutant strain was able to reactivate H2O2-damaged lambda phage. On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities. RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains. The induction of umu operon occurred in UM1 at lower (1/10-1/20) doses of H2O2 than in CSH7. From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E. coli.     

Plasmid modification of radiation and chemical-mutagen sensitivity in Pseudomonas aeruginosa.

The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr+ rec+ (wild-type) genotype and a variety of uvr rec+ type mutants. No protection occurs in a rec A-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plasmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polyA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.     

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.     

Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene.

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H(2)O(2). The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid.

Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H2O2 (a toxic photoproduct) that was selectively lethal for rec and polA1 Escherichia coli mutants. H2O2 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polA1, recA recB, and recA mutants, but were efficiently repaired in wild-type strains. We conclude that H2O2 deoxyribonucleic acid lesions require both the polA+ and recA+ pathways for repair.     

Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli K-12.

We have observed the enzymatic production of deoxyribonucleic acid (DNA) doublestrand breaks in Escherichia coli K12 after ultraviolet irradiation. Doublestrand breaks appeared in wild-type, polA1, recB21, recA, and exrA strains after incubation in minimal medium. THE UVRA6 strain showed no evidence of double-strand breakage under the same conditions. Our data suggest that uvr+ cells, which are proficient in the incision step of excision repair, accumulate double-strand breaks in their DNA as a result of the excision repair process, i.e., arising from closely matched incisions, excision gaps, or incisions and gaps on opposite strands of the DNA twin helix. Furthermore, strains deficient in excision repair subsequent to the incision step (i.e., polA, rec, exrA) showed more double-strand breaks than the wild type strain. The results raise the possibility that a significant fraction of the lethal events in ultraviolet-irradiated, repair-proficient (uvr+) cell may be enzymatically-induced DNA double-strand breaks.     

Dark-Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light III. Effect of rec Mutations on Recovery of Excision-Deficient Mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.     

Oxidative mechanisms of toxicity of low-intensity near-UV light in Salmonella typhimurium.

The exposure of Salmonella typhimurium to environmentally relevant near-UV light stress has been studied by the use of a low-intensity, broad-band light source. The exposure of cells to such a light source rapidly induced a growth delay; after continuous exposure for 3 to 4 h, cells began to die at a rapid rate. The oxidative defense regulon controlled by the oxyR gene was involved in protecting cells from being killed by near-UV light. This killing may be potentiated by the overexpression of near-UV-absorbing proteins. These results are consistent with near-UV toxicity involving the absorption of light by endogenous photosensitizers, leading to the production of active oxygen species. We have shown, however, that one such species, H2O2, is not a major photoproduct involved in killing by near-UV light. Strains lacking alkyl hydroperoxide reductase were more sensitive to near-UV light, indicating that such hydroperoxides may be photoproducts. Near-UV exposure induced sensitivity to high salt levels, indicating that membranes may be a target of near-UV toxicity and a possible source of alkyl hydroperoxides. The demonstration of the inactivation of the heme-containing protein catalase indicates that direct destruction of UV-absorbing macromolecules could be another factor in near-UV toxicity. Cells which have been exposed to near-UV light for long, but sublethal, periods of time (up to 4 h) can recover and resume growth if the UV exposure is stopped but become progressively more sensitive to further stresses, such as H2O2. This result indicates that cells gradually accumulated damage during near-UV exposure until toxic levels were reached.     

Repair of Radiation-Induced Damage in Escherichia coli II. Effect of rec and uvr Mutations on Radiosensitivity, and Repair of X-Ray-Induced Single-Strand Breaks in Deoxyribonucleic Acid

Strains of Escherichia coli K-12 mutant in the genes controlling excision repair (uvr) and genetic recombination (rec) have been studied with reference to their radiosensitivity and their ability to repair X-ray-induced single-strand breaks in deoxyribonucleic acid (DNA). Mutations in the rec genes appreciably increase the radiosensitivity of E. coli K-12, whereas uvr mutations produce little if any increase in radiosensitivity. For a given dose of X-rays, the yield of single-strand breaks has been shown by alkaline sucrose gradient studies to be largely independent of the presence of rec or uvr mutations. The rec(+) cells (including those carrying the uvrB5 mutation) could efficiently rejoin X-ray-induced single-strand breaks in DNA, whereas recA56 mutants could not repair these breaks to any great extent. The recB21 and recC22 mutants showed some indication of repair capacity. From these studies, it is concluded that a correlation exists between the inability to repair single-strand breaks and the radiosensitivity of the rec mutants of E. coli K-12. This suggests that unrepaired single-strand breaks may be lethal lesions in E. coli.     

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detectable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.     

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.     

Recombinational repair of hydrogen peroxide-induced damages in DNA of phage T4

Recently, hydrogen peroxide and its free-radical product, the hydroxyl radical (OH.) have been identified as major sources of DNA damage in living organisms. They occur as ubiquitous metabolic by-products and, in humans, cause several thousand damages in a cell's DNA per day. They are thought to be a major source of DNA damage leading to aging and cancer in multicellular organisms. This raises two questions. First, what pathways are used in repair of DNA damages caused by H2O2 and OH.? Second, a new theory has been proposed that sexual reproduction (sex) evolved to promote repair of DNA in the germ line of organisms. If this theory is correct, then the type of repair specifically available during the sexual process should be able to deal with important natural lesions such as those produced by H2O2 and OH. . Does this occur? We examined repair of hydrogen peroxide damage to DNA, using a standard bacteriophage T4 test system in which sexual reproduction is either permitted or not permitted. Post-replication recombinational repair and denV-dependent excision repair are not dependent on sex. Both of these processes had little or no effect on lethal H2O2 damage. Also, an enzyme important in repair of H2O2-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H2O2 damage to the phage. However, multiplicity reactivation, a recombinational form of repair depending on the sexual interaction of two or more of the bacteriophage, was found to repair lethal H2O2 damages efficiently. Our results lend support to the repair hypothesis of sex. Also the homology-dependent recombinational repair utilized in the phage sexual process may be analogous to the homology-dependent recombination which is widespread in diploid eucaryotes. The recombinational repair pathway found in phage T4 may thus be a widely applicable model for repair of the ubiquitous DNA damage caused by endogenous oxidative reactions.     

Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.     

Interaction between Ustilago Maydis Rec2 and Rad51 Genes in DNA Repair and Mitotic Recombination

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated. rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and γ radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.     

Sensitivity of DNA-repair-deficient strains of Escherichia coli to rifampicin killing

We have analyzed the role of RNA polymerase in DNA repair using the antibiotic rifampicin which binds specifically to the beta subunit of the enzyme. Several DNA-repair-deficient strains such as recA, uvr, and polA, and their isogenic parents were used for this study. All repair-deficient strains were found to be hypersensitive to rifampicin killing. Compared to the isogenic parent strains, recA strains are about 50 times more sensitive and the polA strain is about 100 times more sensitive to rifampicin killing. UvrA and uvrB strains are slightly more sensitive to rifampicin than the wild-type strains. The hypersensitivity of repair-deficient strains to rifampicin killing is totally abolished by the introduction of rifampicin-resistant mutations into these strains. We have examined the effect of rifampicin on RNA and protein synthesis in repair-deficient and -proficient strains. RNA and protein synthesis were found to be inhibited by rifampicin to the same extent among all the strains tested. The results also show that the resumption of DNA synthesis was significantly disrupted in DNA-repair-deficient strains following drug removal. Taken together these results suggest that RNA polymerase plays an essential role in DNA metabolism and such function may be replaced by polA and recA gene products and to a lesser extend by uvrA and uvrB gene products.     

Role of DNA repair genes and an R plasmid in conferring cryoresistance on Pseudomonas aeruginosa

The resistance of Pseudomonas aeruginosa wild-type, uvr, pol and rec strains to ultraviolet (u.v.) light, X-rays and freezing and thawing was determined. An R plasmid, pPL1, which increased resistance of the wild-type uvr, and pol but not rec strains to u.v. light, increased the resistance of only rec and pol mutants to X-rays and freezing and thawing. These findings reinforce the idea of DNA as a target in the organism for freeze-thaw stress and suggest that freeze-thaw-induced DNA damage might be similar to that produced by X-rays but different from that produced by u.v. light.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.