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     

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.     

Excision of pyrimidine dimers by several UV-sensitive mutants of S. pombe

Summary Nine radiation-sensitive mutants of S. pombe showing a variety of phenotypic characteristics were analysed for their ability to excise pyrimidine dimers after ultraviolet irradiation. From earlier studies using indirect parameters, it was expected that some would be excision-deficient. Data reported here show that all the mutants tested, like wild type cells, were able to remove a high percentage of pyrimidine dimers during post-irradiation incubation in several different holding media, but not in saline or phosphate buffer. These mutants included strains showing increased, as well as others which showed decreased, levels of UV-induced mutation frequency relative to that of the wild type at the same total dose.     

Relation between repair mechanisms and induced mitotic recombination after UV irradiation, in the yeast Schizosaccharomyces pombe. Effects of caffeine

Summary Two different pathways A and 1 are known to control the repair of UV lesions in the yeast Schizosaccharomyces pombe. The relation between the UV-induced intergenic mitotic crossing over (MCO) and the repair of prelethal lesions controlled by these pathways were studied in the following strains: UVS1,₁/UVS1,₁, where pathway A acts; UVSA/UVSA where pathway 1 acts, UVS⁺/UVS⁺ (wild type) and UVS1A/UVS1A (double mutant). The analysis of the survival and MCO induction curves, and the comparison, as a function of the dose and as a function of survival, of the MCO induction curves corresponding to the different strains, show that the repair pathway 1 controls a mechanism involving recombination, and that the repair pathway A controls a mechanism which removes prerecombinational lesions. Studies were done with UVS1,₁/UVS1,₁ cells in different physiological conditions affecting the repair efficiency of prelethal lesions (irradiation during the logarithmic growth phase, liquid holding). In all cases the more efficient the repair of prelethal lesions is, the smaller is the recombination inducibility. This is expected if pathway A controls an excision repair mechanism.The effect of the repair inhibitor, caffeine, was studied. It inhibits only the repair of UV prelethal lesions controlled by pathway 1. The involvement of recombination in the repair of UV lesions in UVS⁺/UVS⁺ and UVSA/UVSA cells is also shown by the fact that the sensitization to the lethal effect of UV by caffeine in these strains is correlated with a decrease in UV MCO inducibility. Caffeine has no effect either on the UV survival, or on the MCO inducibility in UVS1,₁/UVS1,₁ cells. It is concluded that it inhibits the recombinational repair pathway and not the excision repair pathway.The MCO induction observed in UVS1/UVS1 and UVS1A/UVS1A cells could be due to the presence of a second recombinational pathway, not sensitive to caffeine. At least a fraction of the prerecombinational lesions would not be prelethal, and they are repairable by the excision repair mechanism.     

A UV-supersensitive mutant in the yeast Schizosaccharomyces pombe

Summary A high UV-sensitive mutant was obtained from a UV-sensitive strain of the yeast Schizosaccharomyces pombe after a mutagenic treatment. By genetic analysis, it was possible to distinguish two independent loci. The double mutant is supersensitive, that is more UV-sensitive than either of the two single mutants. This suggests that the mutations involved interfere with two repair pathways that are, at least partially, independent of each other.Some properties of the two single mutants were studied. These mutants differ notably in their response to caffeine, to liquid-holding, to exposure to visible light after UV irradiation, and in their UV-sensitive during the logarithmic growth phase.Comparison of the properties of the wild-type strain and of the different UV sensitive mutants leads to the conclusion that one repair pathway is used preferentially in the wild-type strain.Abbreviations DRF dose reduction factor - LH liquid holding     

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

Summary Diploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters D_q, D₁₀, D₁, and D₀. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in D_q and D₀ values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (D_q) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.     

Contribution of a caffeine-sensitive recombinational repair pathway to survival and mutagenesis in UV-irradiated Schizosaccharomyces pombe

Summary Cells of wild-type Schizosacharomyces pombe exposed to UV radiation in either G1 or G2 phase show enhanced inactivation of colony-forming ability if plated in the presence of caffeine. This UV-sensitization by caffeine is abolished in both G1 and G2 phase cells by the rad1 mutation; since both caffeine and the rad1 mutation markedly reduce recombinational events, this suggests that a recombinational repair process is active in cells irradiated either in G1 or G2 phase. A prereplicative or sister chromatid exchange recombinational process appears to account for caffeine-sensitive repair of UV-damage in G2 cells (which possess at the time of radiation exposure the duplicated genome necessary for recombination), since caffeine-sensitive repair begins immediately and is completed before resumption of DNA synthesis. In contrast, since caffeine-sensitive repair of UV-damage in G1 cells displays a considerable lag and then occurs concomitantly with DNA synthesis, it appears that G1 cells must acquire a second genome in order to accomplish a caffeine-sensitive recovery process. Since a duplicated genome is required for caffeinesensitive repair, all such repair would seem to involve a recombinational mechanism. In G1 cells the process may be a post-replication recombinational mechanism. Since G2 phase cells are considerably more UV-resistant than G1 phase cells, the prereplicative recombinational process appears to be a much more efficient process for dealing with UV-induced damage than the post-replication mechanism.UV-induced mutagenesis was examined in wildtype and rad mutants using a forward mutation system. Rad mutants which show higher UV-induced mutation rates than wild-type retain UV-sensitization by caffeine (and thus presumably retain the recombinational mechanism). In contrast, rad strains which are relatively UV-immutable compared to wild-type do not possess the caffeine-sensitive UV-repair process. The recombinational process therefore may be the major pathway responsible for UV-induced mutation.AECL Reference No. 6251; NRC Publication No. 16999     

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6-4 photoproducts) in wild-type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision-defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision-defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild-type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.     

Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Recombinational Repair in Schizosaccharomyces pombe: A Role in Maintaining Genome Integrity

Recombinational DNA repair was first detected in budding yeast Saccharomyces cerevisiaeand was also studied in fission yeast Schizosaccharomyces pombeover the recent decade. The discovery of Sch. pombehomologs of the S. cerevisiae RAD52genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered in this review with a focus on comparisons between Sch. pombeand higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.     

The <Emphasis Type="Italic">dds20</Emphasis><Superscript>+</Superscript> Gene Controls a Novel Rad51<Superscript>Sp</Superscript>-Dependent Pathway of Recombinational Repair in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Repair of DNA double-strand break (DSB) is an evolutionary conserved Rad51-mediated mechanism. In yeasts, Rad51 paralogs, Saccharomyces cerevisiae Rad55-Rad57 and Schizosaccharomyces pombe Rhp55-Rhp57 are mediators of the nucleoprotein Rad51 filament formation. As shown in this work, a novel Rad51^Sp-dependent pathway of DSB repair acts in S. pombe parallel to the pathway mediated by Rad51 paralogs. A new gene dds20 ⁺ that controls this pathway was identified. The overexpression of dds20 ⁺ partially suppresses defects of mutant rhp55Δ in DNA repair. Cells of dds20Δ manifest hypersensitivity to a variety of genotoxins. Epistatic analysis revealed that dds20 ⁺ is a gene of the recombinational repair group. The role of Dds20 in repair of spontaneous damages occurring in the process of replication and mating-type switching remains unclear. The results obtained suggest that Dds20 has functions beyond the mitotic S phase. The Dds20 protein physically interacts with Rhp51(Rad51^Sp). Dds20 is assumed to operate at early recombinational stages and to play a specific role in the Rad51 protein filament assembly differing from that of Rad51 paralogs.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 736–745.Original Russian Text Copyright © 2005 by Salakhova, Savchenko, Khasanov, Chepurnaya, Korolev, Bashkirov.     

Effect of caffeine on ozone-sensitivity in Saccharomyces cerevisiae

Summary The addition of 0.1% caffeine to the plating medium markedly reduced the ozone-survival of the wild-type and the rad1 and rad6 mutants of Saccharomyces cerevisiae, whereas no effect was observed in the rad52 mutant. Since, in S. cerevisiae, caffeine has been reported to interfere with the recombinational repair pathway under the control of the RAD52 gene, these results support previous observations suggesting that this pathway is involved in the repair of ozone-induced DNA damage.     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Recombinational repair of alkylation lesions in phage T4

Summary Treatment of bacteriophage T4 by ethyl methanesulfonate (EMS)¹ caused more than a doubling in recombination between two rII markers. The functions of genes 47, 46, 32, 30, uvsX and y are known to be required for genetic recombination, and mutants defective in these genes were found to be more sensitive to inactivation by EMS than wild-type phage. This suggests that a recombinational pathway involving the products of these genes may be employed in repairing EMS induced lethal lesions. Genes 45 and denV are apparently not involved in recombination, and mutants defective in these genes were not EMS-sensitive. Gene 47, 46 and y mutants which were defective in the repair of EMS induced lethal lesions had no detectable deficiency in their ability to undergo EMS-induced mutation. This implies that recombinational repair of EMS lesions does not contribute substantially to EMS mutatenesis. The results obtained here with EMS are in general similar to the results reported in the preceding paper with MNNG, suggesting that the lesions caused by both of these monofunctional alkylating agents may be eliminated by similar recombinational repair processes.     

UV-induced replicating instability in Schizosaccharomyces pombe

A pigmented adenine-requiring strain of S. pombe has been used to study some aspects of the UV-induced replicating instabilities in this yeast. The effect of the repair capacity of a cell on the frequency of such instabilities has been investigated by using three different radiation-sensitive mutants and by studying the influence of caffeine on the frequency of secondary mosaics. Data showed that UV-induced replicating instabilities are not influenced by changes in the repair capacity of the treated cell. Since two of the sensitive mutants used are known to have high spontaneous mutation rates and the third one shows reduced UV-induced mutability, the induction of replicating instabilities differs in this respect both from the induction of spontaneous and UV-induced mutations.     

Effect of protein synthesis inhibition on recovery of UV- and γ-irradiated Schizosaccharomyces pombe from repair inhibition by caffeine

Summary The progress of repair in Schizosaccharomyces pombe may be followed during post-irradiation incubation by measuring, after various intervals, the ability of UV- or -irradiated cells to avoid enhanced lethality when exposed to the repair inhibitor caffeine (Gentner and Werner, 1975). This technique has now been used to investigate the effect of inhibition of protein synthesis on repair of UV- and -irradiation-induced damage in this organism.When protein synthesis was inhibited with cycloheximide in UV-irradiated wild-type cells, only a small amount of recovery from caffeine inhibition occurred; this indicated that post-irradiation protein synthesis was required for repair, and in particular for the recombinational repair pathway, which is a major mechanism for repair of UV damage in this organism.In -irradiated wild-type cells, inhibition of post-irradiation protein synthesis reduced the rate of recovery from repair inhibition by caffeine, but full recovery from caffeine-sensitive damage did occur at longer incubation times. We attribute the reduction in rate to the effect of protein synthesis inhibition on the recombinational repair pathway, because this pathway is known to be involved in the repair of both -ray and UV damage. The recovery that took place at the slower rate must reflect a caffeine-sensitive pathway which is involved only in repair of -ray damage and which does not require post-irradiation protein synthesis for activity.AECL Reference No. 5402     

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

Summary The effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA ⁺ parent bacteria or phenotypically POL⁺ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and c1, indicating that the polA ⁺ and recA ⁺ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.     

The RecRO pathway of DNA recombinational repair in Helicobacter pylori and its role in bacterial survival in the host

Two pathways for DNA recombination, AddAB (RecBCD-like) and RecRO, were identified in Helicobacter pylori, a pathogenic bacterium that colonizes human stomachs resulting in a series of gastric diseases. In this study, we examined the physiological roles of H. pylori RecRO pathway in DNA recombinational repair. We characterized H. pylori single mutants in recR and in recO, genes in the putative gap repair recombination pathway, and an addA recO double mutant that is thus deficient in both pathways that initiate DNA recombinational repair. The recR or recO single mutants showed the same level of sensitivity to mitomycin C as the parent strain, suggesting that the RecRO pathway is not responsible for the repair of DNA double strand breaks. However, H. pylori recR and recO mutants are highly sensitive to oxidative stress and separately to acid stress, two major stress conditions that H. pylori encounters in its physiological niche. The complementation of the recR mutant restored the sensitivity to oxidative and acid stress to the wild type level. By measuring DNA transformation frequencies, the recR and recO single mutants were shown to have no effect on inter-genomic recombination, whereas the addA recO double mutant had a greatly (～12-fold) reduced transformation frequency. On the other hand, the RecRO pathway was shown to play a significant role in intra-genomic recombination with direct repeat sequences. Whereas the recA strain had a deletion frequency 35-fold lower than that of background level, inactivation of recR resulted in a 4-fold decrease in deletion frequency. In a mouse infection model, the three mutant strains displayed a greatly reduced ability to colonize the host stomachs. The geometric means of colonization number for the wild type, recR, recO, and addA recO strains were 6 x 10?, 1.6 x 10?, 1.4 x 10? and 4 x 103 CFU/g stomach, respectively. H. pylori RecRO-mediated DNA recombinational repair (intra-genomic recombination) is thus involved in repairing DNA damage induced by oxidative and acid stresses and plays an important role in bacterial survival and persistent colonization in the host.