Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Relation between liquid-holding recovery, DNA repair, and mitotic recombination in the rad3 mutant of Saccharomyces cerevisiae after treatment with diepoxybutane (DEB)

Summary The rad3 mutant is characterized by a high level of liquid-holding recovery after DEB treatment. The recovery is abolished when the treated cells are postincubated in growth medium, but the effect can be cancelled by suppression of DNA and protein synthesis by specific inhibitors. Alkaline sucrose gradient sedimentation revealed that DEB induces single strand breaks in DNA which are not repaired during post-treatment incubation in growth medium or during LH. Effective repair takes place only when LH is followed by incubation in growth medium. Splitdose treatment applied to test the possible inducibility of repair by LH did not confirm this presumption.In a diploid homozygous for rad3 mutation, DEB induces mitotic inter- and intragenic recombination with very high frequency. Liquid-holding recovery (LHR) was found to be accompanied by an increase in molecular weight of DNA and by a sharp decrease in the frequency of mitotic recombination. The data suggest that recombination events are not involved in LHR pathway.     

Liquid holding recovery in a DEB-Inactivated rad3 strain of Saccharomyces cerevisiae carrying a thermosensitive prt mutation

Summary The mutation prt1-1 with a thermosensitive block in initiation of protein synthesis was introduced into a rad3 strain to study the effect of inhibition of protein synthesis on liquid holding recovery (LHR) from the lethal effects of diepoxybutane (DEB). Liquid holding of the prt1-1rad3 strain under restrictive conditions did not decrease the level of recovery as compared with the permissive temperature. Post-incubation of cells in growth medium under permissive conditions prior to LH resulted in the loss of capacity for LHR, while cells post-incubated under restrictive conditions were fully capable of LHR. The results are interpreted as indicating that protein synthesis during LH is not required for the increase in survival and that the occurrence of protein synthesis prior to liquid holding abolishes the capacity for LHR.     

Study on liquid-holding recovery in DEB-inactivated rad3 mutant of Saccharomyces cerevisiae

Summary Maximal liquid-holding recovery (LHR) of the DEB-treated rad3 mutant occurs at 30° C in buffer supplemented with glucose. Addition of cycloheximide (CHX) to the buffer, the increase in cell density above 2 × 10⁷/ml as well as lowering of temperature during liquid holding (LH) below 27° C decrease considerably the cell capacity for recovery. LHR does not take place at 5° C. No measurable DNA synthesis or degradation occurs in cells held in buffer alone, while addition of 0.02% glucose results in incorporation of radioactivity into DNA both of DEB-treated and control cells. Similarly, protein synthesis was observed only in cultures held in buffer supplemented with glucose. Cells transfered to growth medium directly after treatment complete one round of DNA replication and at least one division cycle, but further DNA replication and cell division are inhibited. Cells placed in growth medium after 5 days LH show an increased rate of DNA replication and cell division. Completion of the first posttreatment round of DNA replication in growth medium abolishes ishes the cell capacity for LHR. DEB treatment results in abnormal cell division of the rad3 mutant, giving colonies consisting of several cells, usually abnormal in shape, held together by common cell walls.     

Responses of radiation-sensitive mutants of Saccharomyces cerevisiae to lethal effects of bleomycin.

Haploid and diploid strains of yeast containing genes conferring radiation-sensitivity were studied under growing and nongrowing experimental conditions for their relative sensitivities to growth-inhibitory effects of bleomycin (BM). The rad1, rad2, rad3, rad4, rad5 (and allelic rev2), rad7, rad10, rad11, rad 12, rad14, rad15, rad16 and rev3 strains exhibited responses similar to normal (Rad+) yeast strains. It is concluded from these findings that the excision-repair function deficient in several of these mutant strains is not important for repair of bleomycin-induced damages in yeast. The sensitive strains contained rad6, rad9, rad18, rad22, rad50, rad51, rad52, rad53, rad54, rad55, rad56, rad57 and rs1. Strains bearing rad8 or rad19 could not be classified unambiguously. With one exception, all rad mutants found very sensitive to BM were sensitive to X-rays, suggesting that some aspect of the repair of BM- and X-ray-induced damages in yeast may be similar. Sensitivities to BM and radiation co-segregated in pedigrees following meiosis, and several BM-resistant revertants isolated from two rad6 mutant strains sensitive to BM, X-rays and UV were cross-resistant to all three agents. These results confirm that the rad mutants were responsible for the cross-sensitivities in the original strains.     

A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae

To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation. When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

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     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

Epistatic Interactions of Radiation-Sensitive (rad) Mutants of CAENORHABDITIS ELEGANS

Six double mutants and a quadruple mutant were derived from four UV radiation-hypersensitive single mutants (rad-1, rad-2, rad-3 and rad-7). Sensitivities of the 11 strains to UV, gamma-radiation and methyl methanesulfonate (MMS) were compared. Of the six double mutants, only the rad-1;rad-2 and rad-3;rad-7 doubles were no more hypersensitive than the most sensitive single mutant to UV-radiation. Thus, rad-1 and rad-2 define one epistasis group, whereas rad-3 and rad-7 define another. Consistent with this model was the observation that rad-1 and rad-2, but not rad-3 and rad-7, were hypersensitive to gamma-radiation. In addition, none of the multiple mutants was more hypersensitive to gamma-radiation than the most sensitive single rad mutant. No synergistic interactions of the rad mutations with respect to MMS sensitivities were observed.     

Saccharomyces cerevisiae RAD53 (CHK2) but not CHK1 is required for double-strand break-initiated SCE and DNA damage-associated SCE after exposure to X rays and chemical agents

Saccharomyces cerevisiae RAD53 (CHK2) and CHK1 control two parallel branches of the RAD9-mediated pathway for DNA damage-induced G(2) arrest. Previous studies indicate that RAD9 is required for X-ray-associated sister chromatid exchange (SCE), suppresses homology-directed translocations, and is involved in pathways for double-strand break repair (DSB) repair that are different than those controlled by PDS1. We measured DNA damage-associated SCE in strains containing two tandem fragments of his3, his3-Delta5' and his3-Delta3'::HOcs, and rates of spontaneous translocations in diploids containing GAL1::his3-Delta5' and trp1::his3-Delta3'::HOcs. DNA damage-associated SCE was measured after log phase cells were exposed to methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), UV, X rays and HO-induced DSBs. We observed that rad53 mutants were defective in MMS-, 4-NQO, X-ray-associated and HO-induced SCE but not in UV-associated SCE. Similar to rad9 pds1 double mutants, rad53 pds1 double mutants exhibited more X-ray sensitivity than the single mutants. rad53 sml1 diploid mutants exhibited a 10-fold higher rate of spontaneous translocations compared to the sml1 diploid mutants. chk1 mutants were not deficient in DNA damage-associated SCE after exposure to DNA damaging agents or after DSBs were generated at trp1::his3-Delta5'his3-Delta3'::HOcs. These data indicate that RAD53, not CHK1, is required for DSB-initiated SCE, and DNA damage-associated SCE after exposure to X-ray-mimetic and UV-mimetic chemicals.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Radiosensitive and mitotic recombination phenotypes of the Saccharomyces cerevisiae dun1 mutant defective in DNA damage-inducible gene expression.

The biological significance of DNA damage-induced gene expression in conferring resistance to DNA-damaging agents is unclear. We investigated the role of DUN1-mediated, DNA damage-inducible gene expression in conferring radiation resistance in Saccharomyces cerevisiae. The DUN1 gene was assigned to the RAD3 epistasis group by quantitating the radiation sensitivities of dun1, rad52, rad1, rad9, rad18 single and double mutants, and of the dun1 rad9 rad52 triple mutant. The dun1 and rad52 single mutants were similar in terms of UV sensitivities; however, the dun1 rad52 double mutant exhibited a synergistic decrease in UV resistance. Both spontaneous intrachromosomal and heteroallelic gene conversion events between two ade2 alleles were enhanced in dun1 mutants, compared to DUN1 strains, and elevated recombination was dependent on RAD52 but not RAD1 gene function. Spontaneous sister chromatid exchange (SCE), as monitored between truncated his3 fragments, was not enhanced in dun1 mutants, but UV-induced SCE and heteroallelic recombination were enhanced. Ionizing radiation and methyl methanesulfonate (MMS)-induced DNA damage did not exhibit greater recombinogenicity in the dun1 mutant compared to the DUN1 strain. We suggest that one function of DUN1-mediated DNA damage-induced gene expression is to channel the repair of UV damage into a nonrecombinogenic repair pathway.     

The Saccharomyces Cerevisiae Rad30 Gene, a Homologue of Escherichia Coli Dinb and Umuc, Is DNA Damage Inducible and Functions in a Novel Error-Free Postreplication Repair Mechanism

Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5.     

Molecular Mechanisms of Pyrimidine Dimer Excision in Saccharomyces cerevisiae: Incision of Ultraviolet-Irradiated Deoxyribonucleic Acid In Vivo

A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD⁺ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.     

MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage

We have characterized the MPH1 gene from Saccharomyces cerevisiae. mph1 mutants display a spontaneous mutator phenotype. Homologs were found in archaea and in the EST libraries of Drosophila, mouse, and man. Mph1 carries the signature motifs of the DEAH family of helicases. Selected motifs were shown to be necessary for MPH1 function by introducing missense mutations. Possible indirect effects on translation and splicing were excluded by demonstrating nuclear localization of the protein and splicing proficiency of the mutant. A mutation spectrum did not show any conspicuous deviations from wild type except for an underrepresentation of frameshift mutations. The mutator phenotype was dependent on REV3 and RAD6. The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin, but not to UV light and X rays. Epistasis analyses were carried out with representative mutants from various repair pathways (msh6, mag1, apn1, rad14, rad52, rad6, mms2, and rev3). No epistatic interactions were found, either for the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity. mph1 slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no effect on X-ray sensitivity was observed. These data suggest that MPH1 is not part of a hitherto known repair pathway. Possible functions are discussed.     

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G2 checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-D (null) mutant is defective in both S and G2 checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G2 checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1, and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G2 checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G2-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G2 arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.