W reactivation is inefficient in repair of the bacterial chromosome

Summary UV-inducible SOS processes associated with W reactivation of phage lambda were studied for their effect on repair of lambda prophage integrated in the bacterial chromosome. For this purpose, lambda cI857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30° to 42°. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30° after UV exposure W reactivation should be fully expressed and should also exert an effect on the bacterial chromosome and the prophage inside it. When heat-induction is delayed until the time at which W reactivation reaches its maximum, a considerable increase in phage survival might then be expected. The results presented in this report show, however, that the delayed induction had only a small effect on the survival of prophage in the wild-type strain (possibly attributable to excision repair) and no detectable effect on prophage in a uvrA strain. From these results we conclude that W reactivation is largely irrelevant to the repair of UV-damaged bacterial DNA.     

Particulate matter inhibits DNA repair and enhances mutagenesis

Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 μg/m³ elevation in long-term exposure to average PM_2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors.     

The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells.

Summary The effect of the ligts-7 mutation on cell survival and the extent of DNA repair after UV (254 nm) irradiation was determined for wild-type and uvrB5 cells of E. coli K-12 at 30° and 42°C. At the restrictive temperature (42°C) the ligts-7 mutation resulted in (i) a decrease in the extent of repair of DNA incision breaks arising during the excision repair process, and (ii) a decrease in the extent of post-replicational repair of gaps in newly-synthesized DNA. These deficiencies in DNA repair correlated with increases in cellular sensitivity to killing by UV radiation. Thus, DNA ligase plays an important role in vivo in both the excision and post-replicational repair processes.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Influence of the yellow Locus on Sensitivity of Drosophila Germ Cells to Chemical Mutagens

The effect of theyellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the sex-linked recessive lethals (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin K; y; y ct v; and y mei-9 ^a) were treated by mutagenic agents and then crossed to Bascfemales. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that theyellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Bascmales were crossed to females of various genotypes including y and y mei-9 ^a ones. The crosses involving y females yielded F₁ progeny with high spontaneous lethality, whereas in F₂, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny F₁. The results obtained using the above two approaches suggest that theyellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.     

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12

Summary Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv ⁺ product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).     

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product

Summary Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.     

The nature of X-ray-induced mutations after recovery in excision repair-deficient (mus-201) Drosophila females

This paper describes the genetic analysis of X-ray-induced mutations at several visible loci (yellow, white, Notch, vermilion and forked) located on the X-chromosome of Drosophila melanogaster after recovery in excision repair-deficient condition (mus-201). A total of 118 mutations observed in 83636 F1 females were analyzed. The white mutations in particular have been investigated at the molecular level. The results show that: (1) the frequency of recovered whole-body mutations is similar or slightly lower in repair-deficient than in repair-proficient condition (respectively 1.5 x 10(-4)/locus/15 Gy and 2.3 x 10(-4)/locus/15 Gy); (2) the frequency of observed mosaic mutations is significantly higher in the repair-deficient condition than in the proficient condition (respectively 2.7 x 10(-4)/locus/15 Gy and 0.9 x 10(-4)/locus/15 Gy); (3) the analysis of F2 male lethal mutations and the cytological analysis of the recovered mutations in the excision repair-deficient condition indicate a decrease in mutations associated with gross chromosomal aberrations (including multilocus deletions); (4) at the molecular level, the spectrum of recovered intragenic mutations is similar after excision-deficient and -proficient repair. These results indicate that excision repair is involved in X-ray-induced DNA damage that is repaired efficiently in the normal repair condition, but bypassed in the excision repair-deficient condition, leading to mosaic mutations. In addition, lesions that apparently cannot be bypassed by DNA replication lead to a decrease in the fraction of mutations due to gross chromosomal aberrations among the whole-body mutations.     

The timing of UV mutagenesis in yeast: continuing mutation in an excision-defective (rad1-1) strain.

Summary Mutation induction by ultraviolet light was studied in excision-defective (rad1-1) strains of Saccharomyces cerevisiae. Information about the timing of mutations in relation to postirradiation DNA replication was obtained. The experimental system involved pedigree analysis of G1 diploid cells and subsequent tetrad analysis of the mitotic segregants to detect mutations.The mutation pattern of rad1-1 strains differed from that of wild type in two respects: (1) few or none of the mutations affected both strands of the DNA, (2) mutations appeared as frequently in the second postirradiation mitotic generation as in the first.The data have led to the following conclusions about the mutation process in excision-defective (rad1-1) yeast: (a) Mutations are not fixed prior to the first postirradiation round of DNA replication. (b) Unexcised thymine dimers persist as mutagenic lesions through repeated postirradiation cell divisions. (c) Heteroduplex repair is involved in the mutation process. (d) Overlapping daughter-strand gaps are not a prerequisite for mutation. (e) The results provide no evidence that error-prone repair in this strain is inducible rather than constitutive. The data also show that (f) all 2-strand mutations (whole-colony mutants) induced in yeast by exposure to low levels of ultraviolet light are associated with excision repair, and that (g) essentially all lesions induced in excision-proficient strains have been excised at the time of the second round of postirradiation DNA synthesis.On leave of absence from the Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh, Scotland EH9 3JN     

The mechanism of untargeted mutagenesis in UV-irradiated yeast

Summary The SOS error-prone repair hypothesis proposes that untargeted and targeted mutations in E. coli both result from the inhibition of polymerase functions that normally maintain fidelity, and that this is a necessary precondition for translesion synthesis. Using mating experiments with excision deficient strains of Baker's yeast, Saccharomyces cerevisiae, we find that up to 40% of cyc1–91 revertants induced by UV are untargeted, showing that a reduction in fidelity is also found in irradiated cells of this organism. We are, however, unable to detect the induction or activation of any diffusible factor capable of inhibiting fidelity, and therefore suggest that untargeted and targeted mutations are the consequence of largely different processes. We propose that these observations are best explained in terms of a limited fidelity model. Untargeted mutations are thought to result from the limited capacity of processes which normally maintain fidelity, which are active during replication on both irradiated and unirradiated templates. Even moderate UV fluences saturate this capacity, leading to competition for the limited resource. Targeted mutations are believed to result from the limited, though far from negligible, capacity of lesions like pyrimidine dimers to form Watson-Crick base pairs.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

Involvement of bacteriophage T4 genes in radiation repair

One interpretation of Ebisuzaki's (1966) observation that the functional survival of certain early phage T4 genes is identical in v⁺ and v -infected cells is that the product of the early gene being studied is essential for the successful completion of excision repair (which is known to be mediated by the v gene). An experiment designed to test this hypothesis is described, with results which fully support the idea. Assuming then that this interpretation is valid, it became possible to determine the involvement in excision repair of a much wider range of early genes by establishing whether or not the v allele affects their functional survival. In addition a comparable series of experiments was performed with phages carrying the u.v.-sensitive y mutation which is known to mediate a quite different type of repair in T4-infected cells.The results indicate that genes 1, 30, 42, 43 and 56 are involved in excision repair, but not genes 32, 41, 43 or 44. All these genes are however involved in y-mediated repair. It appears therefore that this latter repair system (which bears some resemblance to that controlled by the rec genes in bacteria) depends on normal phage DNA synthesis for its completion. However the repair synthesis following the excision of pyrimidine dimers in u.v.-irradiated T4 DNA seems distinct from normal DNA synthesis in that it does not involve certain of the early phage genes, and in particular does not utilize the DNA polymerase coded by gene 43. It is suggested that the polymerase activity associated with this repair synthesis is provided by the bacterial Kornberg polymerase pol I.     

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis

Summary Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.     

Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: The RAD3 system and the RAD51 system

Summary We have studied the role of the excision-repair system and the recombination-repair system in the removal of cross-links and monoadducts caused by furocoumarins plus 360 nm radiation in yeast DNA by neutral and alkaline sucrose gradients and by a fluorometric procedure which detects cross-linked DNA molecules. We found that the excision-repair system, represented by the rad3 mutations, is required both for the removal of monoadducts, causing single-strand break formation, and for the removal of cross-links, causing double-strand break formation. The recombination-repair system, represented by the rad51 mutation, is necessary for double-strand break repair following cross-link removal, but it has no role in the repair of monoadducts.It can be concluded that at least some of the same enzymes are used in yeast for both the excision of pyrimidine dimers and the excision of cross-links or monoadducts caused by furocoumarins plus light. The RAD3 and RAD51 repair systems, which act independently in the repair of UV-induced lesions, are part of a single system for the repair of cross-links.     

Structure and specificity of FEN‐1 from Methanopyrus kandleri

DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG‐like protein Mk0566 from M. kandleri, its biochemical activity and three‐dimensional structure were investigated. Both were found to be more similar to human FEN‐1 than human XPG, suggesting a biological role in replication and long‐patch base excision repair rather than in NER. Proteins 2015; 83:188–194. © 2014 Wiley Periodicals, Inc.     

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

DNA repair in E. coli strains deficient in single-strand DNA binding protein

Summary Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb ^- recA^- and ssb ^- uvr^- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.