The effect of caffeine on repair in Chlamydomonas reinhardtii: II. Interaction of repair systems

Survival following UV-irradiation of the two repair-deficient strains of Chlamydomonas reinhardtii, UVSE5 and UVSE6, was not affected by caffeine. Since caffeine causes increased survival in strains of this organism having normal recombination, these two mutant strains are considered to be recombination-deficient. The double-mutant strains UVSE1–UVSE4, UVSE1–UVSE5, UVSE1–UVSE6, UVSE4–UVSE5, UVSE4–UVSE6 and UVSE5–UVSE6 were isolated. These strains were exposed to UV-irradiation and in all but UVSE4–UVSE5, survival of the double-mutant strain was much lower than for any single-mutant strain. These results indicate that the altered gene products in UVSE1, UVSE5 and UVSE6 mutant strains are associated with different recombination-repair mechanisms.

All double-mutant strains were treated with caffeine following UV-irradiation. In all double-mutant strains containing a mutant USVE4 gene product, recombination repair was increased by caffeine.

On the basis of the data obtained, a scheme is proposed for the involvement of multiple repair systems in repair following UV-irradiation in C. reinhardtii.     

Dark recovery of UV-irradiated phage T1 I. A minor recovery effect whose exclusion permits the study of survival kinetics under presumably repairless conditions

The survival of UV-irradiated phage T1 is much lower in excision repair-deficient than in excision repair-proficient E. coli cells, due to lack of “host cell reactivation” (HCR). An additional decrease in phage survival occurs when repair-deficient (HCR⁻) host cells have been exposed to UV doses from 3000–10 000 erg mm⁻² of 254 nm UV-radiation prior to infection. The observed effect is attributed to loss of a minor phage recovery process, which requires neither the bacterial excision repair nor the bacterial REC repair system. This type of recovery is little affected by caffeine or acriflavine at concentrations that preclude HCR completely. Its full inhibition by UV-irradiation of the cells requires an approximately 8 times larger dose than complete inhibition of HCR.

In heavily preirradiated cells, the T1 burst size is extremely small and multiplicity reactivation is considerably less extensive than in unirradiated cells. Presumably the survival of singly infecting T1 in these cells reflects absence of any type of repair. The observed phage sensitivity and shape of the curve are compatible with the expectation for completely repairless conditions. The mechanism underlying the minor recovery is not known; theoretical considerations make a phage REC repair mechanism seem likely.     

Pleiotropic effects of heterozygosity at the mating-type locus of the yeast Saccharomyces cerevisiae on repair, recombination and transformation

Sexual (MAT a/) and sexual (MAT a/a) strains of the yeast Saccharomyces cerevisiae, which are completely isogenic except at the MAT locus, were compared in their response to ultraviolet radiation. The effects of UV on survival, mitotic intragenic recombination, photoreactivation, and transformation efficiency with UV-irradiated plasmid DNA were examined. The sexual strain had enhanced survival and higher rates of mitotic intragenic recombination compared with the asexual strain. Exposure to visible light subsequent to irradiation increased the survival of both sexual and asexual strains, and decreased their rates of mitotic intragenic recombination. Similar results were obtained by Haladus and Zuk (1980) in their examination of sexual strains homozygous for rad6-1, and wild-type sexuals.

Our sexual strain was also consistently more proficient at transforming plasmid DNA, whether that DNA had been irradiated or not. When pre-irradiated with 25 J/m² of UV, MAT a/ cells transformed more efficiently than MAT a/a cells. When subsequently exposed to light, the ability of these pre-irradiated cells to transform decreased for both strains with increasing irradiation of the plasmid. A smaller decrease in transformation efficiency occurred when cells of both strains were kept in the dark.

When pre-irradiated with 100 J/m², the MAT a/ cells showed a 2-fold increase in their transformation efficiency of both irradiated and unirradiated plasmids by up to 2-fold, a phenomenon not seen in the MAT a/a cells even when pre-irradiated with much higher doses of UV. This increase in transformation efficiency was not, however, seen in the MAT a/ cells when they were exposed to visible light after UV irradiation. These results suggest that cells with the MAT a genotype have a UV-inducible system that increases the efficiency of transformation in the absence of visible light. This increase in transformation is not an induced increase in the repair of plasmid DNA, but rather an increase in the ability of pre-irradiated MAT a/ cells to take up exogenous DNA. MAT a/a cells do not appear to have a similarity inducible system. To the best of our knowledge, this phenomenon has not been previously reported.     

Genetic Effects of Uv Irradiation on Excision-Proficient and -Deficient Yeast during Meiosis

The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD⁺) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m ² to the rad1 strain. Exposure to 25 J/m² had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.     

Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability

Mutation to tryptophan independence after exposure to radiation at the monocrhomatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability. Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA uvrA). Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100. These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair. Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire influence range tested. In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6 and approximately 6.0 with strain WP2. Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr⁺ rec⁺ lex⁺ strains. It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2–4×10⁵ J m²⁻; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system. The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).     

Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.     

Relation Between Survival and Deoxyribonucleic Acid Replication in Ultraviolet-Irradiated Resistant and Sensitive Strains of Escherichia coli B/r

When arabinose-grown Escherichia coli B/r is ultraviolet (UV) irradiated in the logarithmic phase of growth, the dose inactivation curve for both colony formation and deoxyribonucleic acid (DNA) synthesis (based on the relative rates of synthesis) is exponential in nature. When protein synthesis is inhibited before UV-irradiation, both inactivation curves have a large shoulder. Pre-irradiation inhibition of protein synthesis increases considerably the colony-forming ability of a UV-irradiated Hcr(-) and Rec(-) strain of E. coli B/r. However, with the repair-deficient strains, both the shoulder and slope of the survival curve are affected. We investigated the effect of UV irradiation on DNA synthesis in Hcr(-) bacteria and found that pre-irradiation inhibition of protein synthesis increases UV resistance of DNA replication in this strain also. The results suggest that inhibition of protein synthesis before irradiation increases UV resistance in E. coli B/r by a mechanism which is independent of both the excision and recombination repair systems.     

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, ΔuvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation.     

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.     

DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

Effects of 3-(2-alkoxyphenylcarbamoyloxy)chinuclidium chlorides on repair-deficient strains ofChlamydomonas reinhardtii

The effect of five 3-(2-alkoxyphenylcarbamoyloxy)chinuclidium chlorides (alkoxy = butoxy-octyloxy) on survival of a wild-type strain and repair-deficient strains ofChlamydomonas reinhardtii was studied. There was a direct relationship with increased toxic effects in the algal strains as a function of the elongation of the alkyl chain of the alkoxy substituents of the phenylcarbamate acid derivatives. Repairdeficient strains were more sensitive than the wild-type strain. The recombination-deficient strain uvs10 expressed the highest sensitivity to the test agents. This suggests that a gene responsible for recombination repair is involved in an important role in DNA repair of damages induced inC. reinhardtii by the phenylcarbamic esters.     

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12.

The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.     

组蛋白H3K4甲基转移酶复合物亚基Ash2参与裂殖酵母产孢

在氮源缺乏及信息素存在的条件下,裂殖酵母(Schizosaccharomyces pombe)进行减数分裂并完成产孢。在此过程中,信息素介导的MAPK(Mitogen-activated protein kinases)信号通路调控减数分裂相关基因的表达。Spk1是MAPK通路的核心成员,通过蛋白磷酸化的方式激活转录因子Ste11,从而激活mei2⁺、mam2⁺和map3⁺等减数分裂相关基因的表达。尽管组蛋白H3K4甲基化参与基因转录激活、染色质重塑等诸多生物学过程,但其在裂殖酵母产孢过程中的作用并不清楚。文章通过序列比对,发现裂殖酵母Ash2作为H3K4甲基转移酶复合物COMPASS的亚基具有两个保守的结构域,定位于细胞核内参与H3K4的甲基化修饰。ash2⁺的缺失引起裂殖酵母在氮源缺乏时产孢过程的延迟及产孢率下降。ChIP、定量PCR分析结果显示,ash2⁺的缺失降低了spk1⁺编码区H3K4的二甲基化水平,造成spk1⁺mRNA水平的明显下调。在ash2Δ细胞中,虽然ste11⁺的转录水平没有变化,但Ste11的靶基因mei2⁺、mam2⁺和map3⁺的转录水平明显下降。在裂殖酵母中,组蛋白H3K4甲基转移酶复合物COMPASS的亚基Ash2通过调控二甲基化水平修饰从而调节MAPK信号通路,参与裂殖酵母的有性生殖,为建立表观遗传修饰与减数分裂之间的联系提供了新的线索。     

Calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA in U937 cells exposed to tert-butylhydroperoxide: the role of arachidonic acid

Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801-806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca²⁺ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca²⁺ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca²⁺-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca²⁺-dependent activation of phospholipase A₂. Melittin, a potent phospholipase A₂ activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage.     

Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test

Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec⁻) double mutant mei-9^a mei-41^D5 males and Rec⁺ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level, the experiment was repeated with a larval stock consisting of Rec⁺ males and Rec⁺ females under comparable conditions. A preferential killing effect upon Rec⁻ larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons.     

Toxicity of organic acids for repair-deficient strains of Escherichia coli

The wild-type strain and four DNA repair-deficient strains (uvrA6, uvrB5, recA56, and polA1) of Escherichia coli K-12 were treated with acetic acid, lactic acid, and p-aminobenzoic acid at pH 3.5 during their stationary phase of growth. All three acids were highly toxic to the polymerase-deficient strain. The greater sensitivity of the strain carrying the polA1 gene than its isogenic pol+ derivatives suggested that damage caused by acidity requires polA+ gene products for repair.     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.