DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.     

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.     

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.     

Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

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.     

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.     

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.     

Common genes and pathways in the regulation of the mitotic and meiotic cell cycles of Schizosaccharomyces pombe

Summary Cell division cycle mutants defective in G₁, DNA replication or nuclear division were tested for sporulation at semi-restrictive temperatures. In cdc1-7, cdc5-120, cdc17-L16 and cdc18-46 no abnormalities were observed; cdc10-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with a low efficiency; cdc22-M45 was completely defective in meiosis, but could conjugate and formed zygotes with a single nucleus. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc13 were blocked in meiosis II. None of the wee1-50, adh.nim1 ⁺ and win1 ⁺ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis. The meiotic function of cdc13 is TBZ-sensitive and probably exerted downstream of cdc2. Single mutants in cut1 or cut2 did not effect sporulation, whereas the double mutant cut1 cut2 formed two-spored asci. The results demonstrate that the cell division cycle and the meiotic developmental pathway share common genes and regulatory cascades.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

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.     

A role for the yeast cell cycle/splicing factor Cdc40 in the G<Subscript>1</Subscript>/S transition

The CDC40 (PRP17) gene of S. cerevisiae encodes a splicing factor required for multiple events in the mitotic and meiotic cell cycles, linking splicing with cell cycle control. cdc40 mutants exhibit a delayed G₁/S transition, progress slowly through S-phase and arrest at a restrictive temperature in the G₂ phase. In addition, they are hypersensitive to genotoxic agents such as methylmethane sulfonate (MMS) and Hydroxyurea (HU). CDC40 has been suggested to control cell cycle through splicing of intron-containing pre-mRNAs that encode proteins important for cell cycle progression. We screened a cDNA overexpression library and isolated cDNAs that specifically suppress the HU/MMS-sensitivity of cdc40 mutants. Most of these cDNAs surprisingly encode chaperones, translation initiation factors and glycolytic enzymes, and none of them is encoded by an intron-containing gene. Interestingly, the cDNAs suppress the G₁/S transition delay of cdc40 cells, which is exacerbated by HU, suggesting that cdc40 mutants are HU/MMS-sensitive due to their S-phase entry defect. A role of Cdc40p in passage through G₁/S (START) is further supported by the enhanced temperature sensitivity and G₁/S transition phenotype of a cdc40 strain lacking the G₁ cyclin, Cln2p. We provide evidence that the mechanism of suppression by the isolated cDNAs does not (at least solely) involve up-regulation of the known positive START regulators CLN2, CLN3, DCR2 and GID8, or of the large and small essential ribonucleotide reductase (RNR) subunits, RNR1 and RNR2. Finally, we discuss possible mechanisms of suppression by the cDNAs that imply cell cycle regulation by apparently unrelated processes, such as splicing, translation initiation and glycolysis.     

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene

Summary Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc⁺ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc⁺ colonies by UV irradiation. It is postulated that these UV-induced cdc⁺ colonies arise as the result infidelity in DNA replication.     

Multiple phosphorylated forms of the product of the fission yeast cell division cycle gene cdc2 +

Summary The 34 kilodalton protein product (p34) of the cdc2 ⁺ cell cycle control gene of Schizosaccharomyces pombe was expressed in bacteria. Monoclonal antibodies raised against this protein are capable of immunoprecipitating p34^dc2 from yeast lysates. Immunoprecipitates of [³⁵S]methionine- and [³²P]orthophosphate-labeled p34^cdc2 were analyzed by two-dimensional gel electrophoresis. The cdc2 ⁺ gene product is homogeneous in size but resolves into seven species of differing charge. At least four of these species are phosphorylated. Phosphoamino acid analysis reveals that phosphorylation occurs mainly on threonine residues. The pattern of p34 phosphorylation is unaltered at the nonpermissive temperature in strains carrying temperature sensitive alleles of wee1-50 and ran1-114 or in a strain over-producing the ran1 ⁺ gene product.     

Decreased u.v. mutagenesis in an excision-deficient mutant of yeast

The u.v.-sensitive rad4 mutant of yeast was found to decreaseu.v.-induced reversion in the cdc8 and lys2 loci. Survival curvesobtained following u.v. irradiation and u.v.-induced reversionin rad4, cdc8 and cdc8rad4 double mutants show that cdc8 isepistatic to rad4 and belongs to the ‘rad3’ epistasisgroup. A study of u.v.-induced reversion in the thermosensi-tivecdc8 mutant at the restrictive temperature (prevents DNA synthesis)and at the permissive temperature (DNA synthesis takes place)indicates the essential role of DNA replication in u.v.-inducedmutagenesis. ¹To whom correspondence should be addressed     

Evidence that an endo-exonuclease controlled by the NUC2 gene functions in the induction of ‘petite’ mutations in Saccharomyces cerevisiae

Summary Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains. To examine the potential role of this nuclease in the induction of mitochondrial petite mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations. The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain. Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation. First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain. This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to peptite induction by ethidium bromide treatment. Second, a strain having a nuc2 allele that encodes a temperaturesensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures. Petite induction was reduced under restrictive conditions. Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels. Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA. Peptite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment. Taken collectively, these results indicate that the NUC2 gene product functions in the production of mitochondrial petite mutations.     

Mouse p87 wee1 kinase is regulated by M-phase specific phosphorylation

We have cloned a mousewee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50 ^wee1 kinase reported previously. In a cell cycle, the mousewee1 kinase is phosphorylated at M-phase, and anin vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the humanwee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated bycdc2-cyclin B kinase. Furthermore, the activity of thewee1 kinase was reduced by phosphorylation with the mitotic extract which containedcdc2-cyclin B kinase     

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.     

Cyclin-dependent kinase 2 (cdk2) in the murine cdc2 kinaseTS mutant

Kinases of the mammalian cdc2 family including cdk2 (cyclin-dependent kinase 2) are thought to be involved in both the G₂/M transition and DNA replication. To investigate the role of cdc2 kinase and cdk2 in cell cycle progression, murine tsFT210 cells bearing a temperaturesensitive cdc2 mutation were used. These kinases were purified by column chromatography, using a peptide with the consensus phosphorylation site of cdc2 kinase as the substrate. In this mutant, cdc2 kinase activity was temperature sensitive and cdk2 activity was not. At the restrictive temperature, the mutant was only arrested in the G₂ phase and not in the G₂-S phase, suggesting that cdk2 did not compensate for cdc2 kinase at the G₂/M transition but did function at the G₁-S phase. This suggestion was supported by the finding that transfection of cdk2 cDNA did not improve the growth of the mutant cell line at the restrictive temperature, although transfection of cdc2 cDNA did.     

Isolation of a novel type of mutation in the mitotic control of Schizosaccharomyces pombe whose phenotypic expression is dependent on the genetic background and nutritional environment

Summary The major cell cycle control in the fission yeast Schizosaccharomyces pombe acts at entry to mitosis, and involves three previously identified genes cdc2, cdc25 and wee1. The presence of a weel mutation phenotypically suppresses cdc25 mutations. This paper describes the isolation and subsequent analysis of a strain in which the suppression is reversed by the presence of a new mutation, designated win1.1. The mutation causes a slight increase in cell size at division in most genetic backgrounds. However, when combined with a wee1 mutation and cdc25.22, the win 1.1 mutation interacts strongly to generate a novel phenotype: cells are phenotypically cdc during growth on minimal medium but cdc ⁺ when cultured on complex medium. The win1 locus is unlinked to previously identified genes involved in mitosis.     

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.