Repair of MMS-induced DNA double-strand breaks in haploid cells of Saccharomyces cerevisiae, which requires the presence of a duplicate genome.

Summary The formation and repair of double-strand breaks induced in DNA by MMS was studied in haploid wild type and MMS-sensitive rad6 mutant strains of Saccharomyces cerevisiae with the use of the neutral and alkaline sucrose sedimentation technique. A similar decrease in average molecular weight of double-stranded DNA from 5–6x10⁸ to 1–0.7x10⁸ daltons was observed following treatment with 0.5% MMS in wild type and mutant strains. Incubation of cells after MMS treatment in a fresh drug-free growing medium resulted in repair of double-strand breaks in the wild type strain, but only in the exponential phase of growth. No repair of double-strand breaks was found when cells of the wild type strain were synchronized in G-1 phase by treatment with factor, although DNA single-strand breaks were still efficiently repaired. Mutant rad6 which has a very low ability to repair MMS-induced single-strand breaks, did not repair double-strand breaks regardless of the phase of growth.These results suggest that (1) repair of double-strand breaks requires the ability for single-strand breaks repair, (2) rejoining of double-strand breaks requires the availability of two homologous DNA molecules, this strongly supports the recombinational model of DNA repair.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Non-homologous end joining is important for repair of Cr(VI)-induced DNA damage in Saccharomyces cerevisiae

Hexavalent chromium is known to be a potent carcinogen that leads to many different DNA lesions, including DNA-protein crosslinks, and single- and double-strand breaks. In Saccharomyces cerevisiae, DNA double-strand breaks are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ) repair pathways. Here, we show that mutants deficient in NHEJ (yku70Delta, rad50Delta, dnl4Delta, mre11Delta, xrs2Delta) of S. cerevisiae are more sensitive to Cr(VI) toxic effects than wild-type cells. Also, a deletion mutant of SAE2 showed a similar sensitivity to Cr(VI), even though it has no apparent direct role in NHEJ. We also found that double mutants in HR and NHEJ (yku70Delta/rad52Delta, rad50Delta/rad52Delta, dnl4Delta/rad52Delta, mre11Delta/rad52Delta, xrs2Delta/rad52Delta) are synergistically more sensitive to Cr(VI) exposure than any of the single mutants, indicating that both repair pathways are involved in the repair of Cr(VI)-induced lesions. Finally, when the NHEJ mutants were exposed to Cr(VI) under anaerobic growth conditions, Cr(VI) toxicity was suppressed.     

Effectiveness of 1.5 keV aluminium K and 0.3 keV carbon K characteristic X-rays at inducing DNA double-strand breaks in yeast cells

Induction of DNA double-strand breaks in diploid wild-type yeast cells, and inactivation of diploid mutant cells (rad54-3) unable to repair DNA double-strand breaks, were studied with aluminium K (1.5 keV) and carbon K (0.278 keV) characteristic X-rays. The induction of DNA double-strand breaks was found to increase linearly with absorbed dose for both characteristic X-rays. Carbon K X-rays were more effective than aluminium K X-rays. Relative to 60Co gamma-rays the r.b.e.-values for the induction of DNA double-strand breaks were found to be 3.8 and 2.2 for carbon K and aluminium K X-rays respectively. The survival curves of the rad54-3 mutant cells were exponential for both ultrasoft X-rays. For inactivation of rad54-3 mutant cells, the r.b.e.-values relative to 60Co gamma-rays were 2.6 and 2.4 for carbon K and aluminium K X-rays, respectively. The DNA double-strand break data obtained with aluminium K and carbon K X-rays are in agreement with the data obtained for gene mutation, chromosome aberrations and inactivation of mammalian cells, suggesting that DNA double-strand breaks are the possible molecular lesions leading to these effects.     

Radiosensitive strains of Drosophila. XII. Realization of the effect of mutation rad(2)201G1 at the cell and organism levels

Two effects of gamma-rays were studied on radiosensitive mutant rad(2)201G1 and wild type strain rad+ of Drosophila: the rate of radiation-induced chromosome aberrations in somatic cells and lethality of individuals irradiated at different stages of preimaginal development. It has been shown that mutant strain is characterized by the increased rate of chromosome aberrations in somatic cells and lethality of developing flies. Control strain rad+ is characterized by more complicated relationship between the effects analyzed. The results obtained are discussed in connection with the action of rad(2)201G1 gene on repair of genetic damages and with existence of postradiation compensation mechanisms intrinsic in development of multicellular organisms.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Disruption of the<Emphasis Type="Italic">RAD51</Emphasis> gene sensitizes<Emphasis Type="Italic">S. cerevisiae</Emphasis> cells to the toxic and mutagenic effects of hydrogen peroxide

The RAD51 gene was disrupted in three different parental wild-type strains to yield three rad51 null strains with different genetic background. The rad51 mutation sensitizes yeast cells to the toxic and mutagenic effects of H2O2, suggesting that Rad51-mediated repair, similarly to that of RecA-mediated, is relevant to the repair of oxidative damage in S. cerevisiae. Moreover, pulsed-field gel electrophoresis analysis demonstrated that increased sensitivity of the rad51 mutant to H2O2 is accompanied by its decreased ability to repair double-strand breaks induced by this agent. Our results show that ScRad51 protects yeast cells from H2O2-induced DNA double-strand breakage.     

Normal DNA ligase activity in a gamma-ray-sensitive Chinese hamster mutant

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by gamma-irradiation during the sensitive G1--early-S period and somewhat deficient in repair of gamma-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5'-phosphate-3'-hydroxyl breaks in double-stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G1-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.     

The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae.

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.     

Normal DNA ligase activity in a γ-ray-sensitive Chinese hamster mutant

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by γ-irradiation during the sensitive G₁-early-S period and somewhat deficient in repair of γ-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5′-phosphate-3′-hydroxyl breaks in double stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G₁-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.     

Characterization and quantitation of DNA strand breaks requiring recA-dependent repair in X-irradiated Escherichia coli

The repair of X-ray-induced DNA single-strand breaks was studied after the completion of growth-medium-independent repair in Escherichia coli K-12. A comparison of the sedimentation of DNA from bacteriophages T2 and T7 was used to test the accuracy of our alkaline and neutral sucrose gradient procedures for determining the molecular weight of bacterial DNA. The repair of DNA single-strand breaks by cells incubated in buffer occurred by two processes. About 85% of the repairable breaks were resealed rapidly (t1/2 = less than 6 min), while the remainder were resealed slowly (t1/2 = approximately 20 min). After the completion of the repair of DNA single-strand breaks in buffer, about 80% of the single-strand breaks that remained were found to be associated with DNA double-strand breaks. The subsequent resuspension of cells in growth medium allowed the repair of both DNA single- and double-strand breaks in wild-type but not in recA cells. Thus the recA-dependent, growth-medium-dependent repair of DNA single-strand breaks is essentially the repair of DNA double-strand breaks.     

Rad50 is not essential for the Mre11-dependent repair of DNA double-strand breaks in Halobacterium sp. strain NRC-1

The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), and gamma radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.     

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.     

Split-dose recovery is due to the repair of DNA double-strand breaks

DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad 54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break.     

Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.     

Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways.

Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.     

Roles of <Emphasis Type="Italic">Saccharomyces cerevisiae RAD17</Emphasis> and <Emphasis Type="Italic">CHK1</Emphasis> checkpoint genes in the repair of double-strand breaks in cycling cells

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background. Nelson Bracesco and Ema C. Candreva have contributed equally to this article.     

UV response of the temperature-conditional rad 54 mutant of the yeast Saccharomyces cerevisiae

The survival of the yeast mutant rad 54-3, which is temperature-conditional for the repair of double-strand breaks, was measured after exposure to UV-light (254 nm) and incubation at 23 degrees C and 36 degrees C. It was found that survival was drastically reduced with incubation at the restrictive temperature. Temperature-shift experiments indicated that repair of UV-induced damage which is controlled by the rad 54 gene proceeds with a half-value-time of about 7 h.     

The Saccharomyces Cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G(1) and G(2) or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.     

Deoxyribonucleic acid strand breaks during drying of Escherichia coli on a hydorohobic filter membrane.

Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures. A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above. Sucrose gradient studies showed that single- and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above. These results were observed in all cells rehydrated with 0.03 M tris (hydroxymethyl) aminomethane-hydrocholoride buffer solution at 0 or 37 degrees C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution. Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry. Removal of unfreezable water of cells in the drying, therfore, might induce deoxyribonucleic acid strand breaks.