Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA

Summary Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before. This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast. There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5-fold, and where rad1-1 and rad6-1 survival increased 2-fold. It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA. It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover.     

Cisplatin-modification of DNA repair and ionizing radiation lethality in yeast,Saccharomyces cerevisiae

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.     

Expression of the cloned endo-1,3-1,4-β-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae

Summary A cloned endo-1,3-1,4--glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fragment. This gene has been sub-cloned in the yeast LEU2 vector pJDB207 to produce a hybrid plasmid designated pEHB9. pEHB9 has been transformed to S. cerevisiae and shown to direct the synthesis of an endo-1,3-1,4--glucanase in yeast. The -glucanase activity was low and could only be detected in crude cell extracts of yeast harbouring pEHB9.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes

The green alga Chlamydomonas reinhardtii is a convenient model organism for the study of basic biological processes, including DNA repair investigations. This review is focused on the studies of DNA repair pathways in C. reinhardtii. Emphasis is given to the connection of DNA repair with other cellular functions, namely the regulation of the cell cycle. Comparison with the results of repair investigations that are already available revealed the presence of all basic repair pathways in C. reinhardtii as well as special features characteristic of this alga. Among others, the involvement of UVSE1 gene in recombinational repair and uniparental inheritance of chloroplast genome, the specific role of TRXH1 gene in strand break repair, the requirement of PHR1 gene for full activity of PHR2 gene, or encoding of two excision repair proteins by the single REX1 gene. Contrary to yeast, mammals and higher plants, C. reinhardtii does not appear to contain the ortholog of RAD6 gene, which plays an important role in DNA translesion synthesis and mutagenesis. Completed genome sequences will be a basis for molecular analyses allowing to explain the differences that have been observed in DNA repair of this alga in comparison with other model organisms.     

Interaction of excision repair gene products and mitotic recombination functions in yeast

We have tested the ability of mutants of three additional genes in the excision repair pathway of Saccharomyces cerevisiae to suppress the hyper-recombination and rad52 double-mutant lethality phenotypes of the rad3-102 (formerly rem1-2) mutation. Such suppression has previously been been observed with mutant alleles of RAD1 and RAD4. We had hypothesized that the rad3-102 mutation created elevated levels of DNA lesions which could be processed by the products of the RAD1 and RAD4 genes into recombinogenic double-strand breaks requiring the RAD52 product for repair. In this report, we show that the RAD2, RAD7, and RAD10 genes are also necessary for this processing. We discuss our observations of varying levels of mitotic crossingover in Rem^- rad double-mutant strains.     

Cloning and identification of a DNA fragment coding for the sup1 gene of Saccharomyces cerevisiae

Summary A plasmid, pYsup1-1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR).     

Synergism between yeast nucleotide and base excision repair pathways in the protection against DNA methylation damage

The treatment of cells with simple DNA methylating agents such as methyl methanesulfonate (MMS) results in genotoxic lesions, including 3-methyladenine which blocks DNA replication. All the organisms studied to date contain an alkylation-specific base excision repair pathway. In the yeast Saccharomyces cerevisiae, the base excision repair pathway is initiated by a Mag1 3-methyladenine DNA glycosylase that removes the damaged base, followed by the Apn1 apurinic/apyrimidinic endonuclease which cleaves the DNA strand at the abasic site for subsequent repair and synthesis. Several nucleotide excision repair pathway mutants display only slightly increased sensitivity to killing by MMS, indicating that nucleotide excision repair per se does not play a major role in the repair of DNA methylation damage. However, mag1 and apn1 mutants that are also defective in nucleotide excision repair are extremely sensitive to MMS-induced killing and the effects are synergistic. These observations suggest that nucleotide excision repair and alkylation-specific base excision repair provide alternative pathways for the repair of DNA methylation damage. In addition to their role in nucleotide excision repair, Rad1 and Rad10 form a complex that is involved in recombination repair. It was found that the apn1 rad1 and apn1 rad10 double mutants have a growth defect and are significantly more sensitive to MMS killing than apn1 rad2 and apn1 rad4 double mutants in a gradient plate assay. Furthermore, the apn1 rad1 double mutant increased both the spontaneous and MMS-induced mutation frequency. Thus, the recombination repair defects of rad1 and rad10 may confer an additional synergistic effect when combined with the apn1 mutation. Received: 8 September 1997 / 13 November 1997     

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.     

Physical and functional characterization of the cloned lysl + gene of Schizosaccharomyces pombe

The α-aminoadipate pathway for the biosynthesis of lysine is present in yeast and other higher fungi. The lys2 and lys5 mutants of Saccharomyces cerevisiae as well as the lys1– and lys7 –mutants of Schizosacharomyces pombe are blocked at the α-aminoadipate reductase step of this pathway. The cloned lys1 + gene in the plasmid pLYS1 isolated from a S. pombe genomic library complemented lys1– mutant of S. pombe. The cloned LYS2 gene in the plasmid YEp620 and the LYS5 gene in the plasmid pSC5 of S. cerevisiae exhibited heterologous complementation of lys1– and lys7– mutants, respectively, of S. pombe. The homologous lys1 + transformed cells exhibited five fold higher α-aminoadipate reductase activity while the heterologous lys1 + and lys7 + transformed cells exhibited much less activity than the the wild type cells. The DNA insert of the plasmid pLYS1 was determined to be 16.7 kb long and the lys1 + gene has been subcloned within a 9.1 kb Clal-Clal DNA insert of the recombinant plasmids pLYS1B and pLYS1C. The restriction pattern for 12 enzymes of the 9.1 kb DNA insert, (Apal, Aval, BamHI, Clal, EcoRI, EcoRV, HindIII, Hpal, Pstl, Pvull, Sphl, and Xbal), exbibited no obvious similarity to that of the LYS2 gene of S. cerevisiae. A 1.7 kb EcoRI-HindIII DNA fragment of pLYS1B and pLYS1C complemented the lys1-131 mutation in an integrative transformation. Although the lys1 + gene of S.pombe is isofunctional to the LYS2 gene of S. cerevisiae, the restriction sites, and expression of these two genes exhibited considerable divergence.     

Cloning of a yeast dihydrofolate reductase gene in Escherichia coli

Summary The dihydrofolate reductase gene of Saccharomyces cerevisiae has been isolated by selection of trimethoprim resistant Escherichia coli transformed with a gene bank of yeast DNA in plasmid pBR322. From 9.2 kilobase pair BamHI DNA fragment this gene has been localized to a 1.76 kb fragment, the restriction map of which appears different from those reported for the E. coli and the mouse dihydrofolate reductase genes.The enzyme encoded by the chimeric plasmid was established as yeast dihydrofolate reductase by its sensitivity to antifolates in vivo through growth studies and in vitro by enzyme assay. Since, the expression of this gene occurs independent of its orientation within the chimeric plasmid, the 1.76 kb fragment may contain functional regulatory sequences in addition to the structural sequences for yeast dihydrofolate reductase.This work was carried out in part at Merck & Co., Rahway, New Jersey, USA and at Southern Biotech, Inc., Tampa, Florida. USA     

Repair of ultraviolet light damage in Saccharomyces cerevisiae as studied with double- and single-stranded incoming DNAs

Summary Purified double- and single-stranded DNAs of the autonomously replicating vector M13RK9-T were irradiated with ultraviolet light (UV) in vitro and introduced into competent whole cells of Saccharomyces cerevisiae. Incoming double-stranded DNA was more sensitive to UV in excision repair-deficient rad2-1 cells than in proficient repair RAD ⁺ cells, while single-stranded DNA exhibited high sensitivity in both host cells. The results indicate that in yeast there is no effective rescue of UV-incoming single-stranded DNA by excision repair or other constitutive dark repair processes.     

Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1‐dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose‐dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild‐type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

Summary We have developed a system for assaying pyrimidine dimers in the 2 m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m^–2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.     

Search for DNA repair pathways in Drosophila melanogaster

The knowledge about the existence of different pathways for the repairing of DNA lesions has made possible a better understanding of mutation processes. The double mutant method has been shown to be useful for grouping rad mutants in yeast. Through this method, three different groups of repair mechanisms were found: (a) RAD3 group corresponding to the excision repair of UV lesions, (b) RAD6 group corresponding to the translesion type of post-replication repair and, (c) RAD52 group corresponding to the recombination type of post-replication repair. In this work, a search for a classification of Drosophila mus mutants in groups analogous to yeast RAD groups is done. Information obtained by double mutant studies was integrated with that obtained by biochemical, recombination, DNA damaging agent sensitivity and mutation studies. The following groups were found: (a) group of mei9 and mus201, analogous to RAD3, (b) group of mei41 and mus302 analogous to RAD52 and, (c) group of mus104 and mus101 analogous to RAD6. In addition, there are mutants that belong to a group corresponding to pre-replication repair of MMS lesions such as mus103, mus306 and mus207. As a peculiarity of Drosophila, it was found that interaction between pre- and post-replication repair mechanisms is indifferent and not synergistic as was found in yeast. A possible explanation could be a weaker control of post-replication repair mechanisms in Drosophila than in yeast. It is expected that this research could help for a better understanding of repair mechanisms in complex organisms.     

Interactions among mutations affecting spontaneous mutation,mitotic recombination,and DNA repair in yeast

The mutant alleles mms9-1, mms13-1, or mms21-1 of Saccharomyces cerevisiae confer pleiotropic effects, including sensitivity to the alkylating agent methyl methanesulfonate, elevations in spontaneous mutation and mitotic recombination, defects in meiosis, and cross-sensitivity to radiation. We constructed double-mutant strains containing an mms mutation and a defect in either excision repair, mutagenic repair, or recombinational repair and measured the levels of spontaneous mutation and mitotic reombination. Double mutants lacking excision repair show elevations in spontaneous mutation but with predominantly unchanged levels of mitotic recombination. RAD52 function was required for the expression of the hyper-recombination phenotype of the mms9-1, mms13-1, and mms21-1 alleles; double mutants displayed the very low recombination levels characteristic of rad52 mutants. Phenotypes of double mutants containing one of the mms alleles and either of the hyper-recombination/mutator rad6-1 or rad3-102 alleles suggest that the mutagenic lesions in mms strains may not be identical to the recombinogenic lesions.     

Genetic control of plasmid DNA double-strand gap repair in yeast,Saccharomyces cerevisiae

Summary The repair of double-strand gaps (DSGs) in the plasmid DNA of radiosensitive mutants of Saccharomyces cerevisiae has been analyzed. The proportion of repair events that resulted in complete plasmid DNA DSG recovery was close to 100% in Rad⁺ cells. Mutation rad55 does not influence the efficiency and preciseness of DSG repair. The mutant rad57, which is capable of recombinational DNA DSB repair, resulted in no DSG recovery. Mutation rad53 substantially inhibits the efficiency of DSG repair but does not influence the precision of repair. Plasmid DNA DSG repair is completely blocked by mutations rad50 and rad54.     

Identification and isolation of the cytochrome oxidase subunit II gene in mitochondria of the yeast Hansenula saturnus

Summary Mitochondrial DNA from the petite negative yeast Hansenula saturnus has been isolated and sized by digestion with restriction enzymes. The size of the mitochondrial genome is approximately 47 kb. The gene for subunit II of cytochrome oxidase was localized in the genome by Southern blotting using a [³²P]-labeled probe containing the subunit II gene of the yeast Saccharomyces cerevisiae. The probe hybridized to a 1.7 kb HindIII-BamHI fragment under stringent conditions (65°C), indicating a high degree of homology between the S. cerevisiae and H. saturnus mitochondrial DNA fragments. The 1.7 kb fragment from H. saturnus was cloned into pBR322 and physically mapped. The map was used to obtain the nucleotide sequence of the subunit II gene (Lawson and Deters presented in the accompanying paper).     

Gene PSO5 of Saccharomyces cerevisiae,involved in repair of oxidative DNA damage,is allelic to RAD16

The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H₂O₂ and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.     

The repair of DNA methylation damage in Saccharomyces cerevisiae

 The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways. Received: 13 June/24 July 1996