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.     

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.     

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.     

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.     

The C-terminal part of the CDC25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-CDC25 protein

The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity. A chimera between the 3 regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal CDC25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the CDC25 and the SDC25 gene products are structurally and functionally similar. The CDC25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.     

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.     

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.     

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions

Summary Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cells remain vegetative at the restrictive temperature (35°C) in — N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25°C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25°C dedifferentiate into vegetative cells under N-starvation conditions after transfer to 35°C, but differentiate again into gametes at 25°C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the matingtype locus; the dif-1 phenotype cosegregates with a conditional flagellales phenotype expressed in both +N and-N medium at the restrictive temperature.     

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae

Summary We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MAT rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36°C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36°C for both mitotic recombination and sporulation.     

Extragenic revertants of rad50, a yeast mutation causing defects in recombination and repair

Summary The RAD50 gene in yeast is required for recombination-repair (i.e., the double strand break repair pathway) in mitosis, and for meiotic recombination and sporulation. Both of these processes are complex and seem likely to require a relatively large number of gene products. In order to help define other genes required for recombination and repair processes in yeast, we have isolated extragenic revertants of rad50-4 which restore the ability to grow in the presence of MMS. Evidence from segregation indicates the extragenic revertants fall into at least five loci. Two of them reduce sporulation and spore viability at high temperature; another mutation confers a spontaneous hyperrec phenotype on mitotic cells. Thus, at least three revertants are candidates for mutations which affect recombination functions.     

Cloning and integrative deletion of the RAD6 gene of Saccharomyces cerevisiae

Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad⁺ levels of resistance to U.V., MMS and -rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids.Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.     

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.     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

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.     

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.     

Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe

Background

The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts. Six MCM subtypes have been identified in various species. These MCM proteins are thought to bind each other to make a heteromeric complex. The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication.

Results

The suppressor mutant of nda4 was isolated and the mutant gene was named sna41. The sna41-912 mutant demonstrated the ts phenotype, with an elongated cell shape at the restrictive temperature. Cells with 1C DNA content accumulated 2 h after shifting up to the restrictive temperature. This result suggests that sna41 is also involved in DNA replication. The sna41 genomic clone was isolated by a complementation of the ts phenotype of the mutant strain and was sequenced. The sna41 gene encodes a protein of 638 amino acids, which has low homology with CDC45 in S. cerevisiae. The gene disruption analysis showed that sna41 gene is essential for viability.

Conclusions

The S. pombe sna41 mutation suppresses the nda4-108 mutation. Sna41 is involved in DNA replication and may play some roles in the regulation of DNA replication by the MCM proteins.

     

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3′ end-processing

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA^Asp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3 end-processing of the tRNA^Asp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3 end-processing. One such suppressor mutation was further characterized: it restores tRNA^Asp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.     

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.     

uvsI mutants defective in UV mutagenesis define a fourth epistatic group of uvs genes in Aspergillus

Three UV-sensitive mutations of A. nidulans, uvsI, uvsJ and uvsA, were tested for epistatic relationships with members of the previously established groups, here called the UvsF, UvsC, and UvsB groups. uvsI mutants are defective for spontaneous and induced reversion of certain point mutations and differ also for other properties from previously analyzed uvs types. They are very sensitive to the killing effects of UV-light and 4-NQO (4-nitro-quinoline-N-oxide) but not to MMS (methylmethane sulfonate). When double-and singlemutant uvs strains were compared for sensitivity to these three agents, synergistic or additive effects were found for uvsI with all members of the three groups. The uvsI gene may therefore represent a fourth epistatic group, possibly involved in mutagenic repair. On the other hand, uvsJ was clearly epistatic with members of the UvsF group and fitted well into this group also by phenotype. The uvsA gene was tentatively assigned to the UvsC group. uvsA showed epistatic interactions with uvsC in all tests, and like UvsC-group mutants is UV-sensitive mainly in dividing cells. However, the uvsA mutation does not cause the defects in recombination and UV mutagenesis typical for this group.