Role of the CDC8 gene in the repair of single strand breaks in DNA of the yeast Saccharomyces cerevisiae

Summary It has been found that the repair of single strand breaks is defective in the DNA replication mutants cdc8-1 and cdc8-3 of Saccharomyces cerevisiae both in permissive (23°C) and restrictive conditions (36°C). In permissive conditions we observed a significant delay in single strand break repair in a diploid strain HB7 (cdc8-1/cdc8-1), as compared with the wild-type strain. Under restrictive conditions no repair was observed, but rather degradation of MMS-damaged DNA occurred. It has been also found that the repair of single strand breaks in yeast is inhibited by cycloheximide but not by hydroxyurea.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

Evidence that the single CDC8 gene which encodes thymidylate kinase is involved in DNA replication,recombination and mutation in yeast

Summary The conditional cdc8 mutant is known to be defective, under restrictive conditions, in the elongation of DNA during synthesis. In yeast the CDC8 gene encodes thymidylate kinase. We show here that UV-induced gene conversion and gene mutation events require the participation of this CDC8 gene. Thus, the same thymidylate kinase is incolved both in DNA replication and in UV-induced gene conversion and gene mutation in yeast.     

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, δ and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase δ, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase δ and a deletion in the REV3 gene had a complete repair defect, both at permissive (23°C) and restrictive (38°C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase ɛ, nor deletion in the DNA polymerase IV (β₇₀) gene, caused defective repair. The data obtained suggest that DNA polymerases δ and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis. Received: 18 November / 10 December 1996     

Single-strand scissions of nuclear yeast DNA occur without meiotic recombination

Summary In meiotic cells of Saccharomyces cerevisiae, reduction in molecular weights of DNA in alkaline sucrose gradients is observed concomittantly with premeiotic DNA replication and with commitment to recombination. Following the completion of the latter processes, higher molecular weights are obtained. These single-stranded breaks are found in both old and newly synthesised strands. Similar scissions in DNA are also found in a temperature-sensitive mutant (cdc40/cdc40), which does not undergo commitment to recombination at the restrictive temperature, and in vegetative wild type cells that were previously exposed to sporulation medium. The suggestion that these scissions are the physical manifestation of commitment to recombination is therefore rejected.     

The PSO4 gene of S. cerevisiae is important for sporulation and the meiotic DNA repair of photoactivated psoralen lesions

We have evaluated the effect of the Saccharomyces cerevisiae pso4-1 mutation in sporulation and DNA repair during meiosis. We have found that pso4-1 cells were arrested in an early step of meiosis, before premeiotic DNA synthesis, and hence did not produce spores. These results suggest that the PSO4 gene may act at the start point of the cell cycle, as do some SPO and CDC genes. The pso4-1 mutant cells are specifically sensitive to 8-MOP- and 3-CPs-photoinduced lesions, and are found to be severely affected in meiotic recombination as well as impaired in the mutagenic response, as previously described for mitosis. This means that the PSO4 gene is important for the repair 8-MOP-photoinduced lesions, mainly double-strand breaks, and the processing of these lesions into recombinogenic intermediates.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

Exposure of cdc8 homozygous diploids of Saccharomyces cerevisiae to nonpermissive temperature leads to an increase in mitotic conversion frequencies

Summary In a diploid strain homozygous for the cdc8-1 mutation, a block in DNA synthesis caused by restrictive temperature resulted in a significant increase in the frequency of intragenic recombination at the HOM2 locus. Under restrictive conditions, incorporation of radioactivity into DNA was reduced to 2% of the control and alkaline sucrose gradient centrifugation revealed that only short DNA fragments were synthesized. There was no considerable fragmentation of template DNA during incubation of cdc8-1 strains under restrictive conditions.     

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     

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     

Chemical mutagenesis and DNA synthesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

Summary Incubation of cdc8 mutants of the yeast Saccharomyces cerevisiae in YPD under permissive conditions, when DNA replication is taking place, prior to transfer to restrictive conditions, strongly stimulates induction of cdc ⁺ colonies of ethyl methane sulphonate (EMS)- and methyl methane sulphonate (MMS)-treated yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). After diepoxybutane (DEB) treatment, both the induction of cdc ⁺ colonies and their stimulation after incubation in YPD under permissive conditions is low. The results obtained show that stimulation of induction of cdc ⁺ colonies under permissive conditions occurs not only after UV-treatment, but also after treatment with such mutagens as EMS and MMS.     

The mcm2-1 mutation of yeast causes DNA damage with a RAD9 requirement for repair

The minichromosome maintenance mutation, mcm2-1, has been found to synthesize damaged DNA at 35°C. Growth at this temperature rendered the mutant strain more sensitive to killing by ultraviolet irradiation. DNA damage could also be detected by pulsed-field gel electrophoresis, where a higher fraction of the DNA loaded was retained in the inserts at the wells. During the exponential phase of growth at this temperature about 50% of the cells had large buds, with the nucleus at or near the neck of the bud in most cases. The incorporation of the rad9 deletion in the mcm2-1-carrying strain caused a reduction in the percentage of large-budded cells and a moderate loss of cell viability. The results are consistent with mcm2-1 causing DNA damage leading to the arrest of cells in the S/G₂ phase of the cell cycle, which was partially dependent on the RAD9 gene product.     

The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase

Summary DNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD ⁺ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD ⁺ strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m⁵C residues.     

Suppression of a DNA polymerase δ mutation by the absence of the high mobility group protein Hmo1 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The deletion of the gene encoding the high mobility group protein Hmo1 suppresses the growth retardation of the DNA pol δ mutation, pol3-14, at the restrictive temperature. pol3-14 mutant cells undergo cell cycle arrest, and hmo1Δ alleviates the arrest permitting continual division of the double mutant. Bypass of cell cycle control occurs with an increased rate of mutation. Both pol3-14 and hmo1Δ are mutators and their combination provokes a synergistic rate of CAN1 mutations. RAD18 controls branches of DNA repair pathways and its deletion also suppresses pol3 mutations. Comparing hmo1Δ and rad18Δ suppression of pol3-14 shows that while both require the presence of RAD52-mediated repair, their suppression is independent in that both can suppress in the presence of the other. We conclude that hmo1Δ suppression of pol3-14 occurs by a mechanism whereby normal controls on DNA integrity are breached and lesions flow into RAD52-mediated repair and error-prone pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reassessing the genotoxic potential of 8-MOP + UVA-induced DNA damage in the yeast Saccharomyces cerevisiae

Summary Two different UVA irradiation systems were initially biologically calibrated with two haploid yeast strains proficient and deficient, respectively, in nucleotide excision repair. The number of DNA lesions introduced into the cell's genome by the photoactivated bifunctional furocoumarin 8-MOP was then calculated by means of the applied UVA exposure doses. At LD₃₇ the repair-proficient wild type had about 14 ICL and 34 furan-side monoadducts in its DNA, while doubly blocked repair mutant rad3-12 pso1-1 had 2 ICL and 3 monoadducts. Locus-specific reversion of lys1-1 followed two-hit kinetics in the repair-proficient wild type and one-hit kinetics in an excision-deficient rad2-20 mutant, as would be expected if ICL was the main type of mutagenic lesion in the wild type and monoadducts the main mutagenic lesion type in the excision-deficient strain. Quantitative comparison of 8-MOP + UVA-induced ICL with those induced by bifunctional mustard revealed the former to have a much higher genotoxicity.Abbreviations UVA Ultraviolet irradiation of 365 nm - 8-MOP 8-methoxypsoralen - ICL interstrand cross-link(s)     

Isolation and characterization of three mutants with increased sensitivity to photoactivated 3-carbethoxypsoralen in Saccharomyces cerevisiae

The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxy-psoralen define three novel recessive mutant alleles pso-5-1, pso6-1, and pso7-1. Their cross-sensitivity to UV_254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions. None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV_254nm.     

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.     

Heat shock changes the response of the pso3 mutant of Saccharomyces cerevisiae to 8-methoxypsoralen photoaddition

A putative tolerance, induced by heat shock (HS), to the lethal and mutagenic effects of 8-methoxypsoralen (8-MOP) photoaddition and hyperthermia was analyzed in Saccharomyces cerevisiae using the wild-type strain N123 and the isogenic DNA repair-deficient mutant pso3-1. In wild-type cells, the HS (38°C for 1 h) did not modify either the survival or the mutation frequency observed after 8-MOP photoaddition, even though it conferred protection against the lethal effect of hyperthermia (50°C). In the pso3-1 mutant, HS induced an increase of the survival, and a decrease of the mutation frequency, after 8-MOP photoaddition and it also protected against the lethal effect of hyperthermia. The responses induced by HS were specific for 8-MOP photoaddition, since they were not observed after 254 nm ultraviolet-light damage. These results indicate that the protection conferred by HS depends of the type of lesion, and operates through the induction of different repair processes. In the pso3-1 mutant, HS could channel the repair intermediates to and error-free repair pathway.     

λ-Radiation-induced irreparable damage to DNA of HeLa cells

Exposure of HeLa cells to λ-radiation at 0.1 Gy and then at 5 Gy reduces their ability to repair double-strand DNA breaks to a greater extent than irradiation with a single dose of 5 Gy. Modifying effects of 0.1 Gy on double-strand DNA breaks and on cell survival are observed after irradiation during logarithmic but not stationary phase of growth. Primary λ-induced irreparable double-strand breaks correlates with cell survival regardless the irradiation regime. It is suggested that such a damage is primarily responsible for reproductive death of HeLa cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 7, pp. 53–56, July, 1997