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In vitro, the protein complex Chromatin Assembly Factor-I (CAF-I) from human or yeast cells deposits histones onto DNA templates after replication. In Saccharomyces cerevisiae, the CAC1, CAC2, and CAC3 genes encode the three CAF-I subunits. Deletion of any of the three CAC genes reduces telomeric gene silencing and confers an increase in sensitivity to killing by ultraviolet (UV) radiation. We used double and triple mutants involving cac1Delta and yeast repair gene mutations to show that deletion of the CAC1 gene increases the UV sensitivity of cells mutant in genes from each of the known DNA repair epistasis groups. For example, double mutants involving cac1Delta and excision repair gene deletions rad1Delta or rad14Delta showed increased UV sensitivity, as did double mutants involving cac1Delta and deletions of members of the RAD51 recombinational repair group. cac1Delta also increased the UV sensitivity of strains with defects in either the error-prone (rev3Delta) or error-free (pol30-46) branches of RAD6-mediated postreplicative DNA repair but did not substantially increase the sensitivity of strains carrying null mutations in the RAD6 or RAD18 genes. Deletion of CAC1 also increased the UV sensitivity and rate of UV-induced mutagenesis in rad5Delta mutants, as has been observed for mutants defective in error-free postreplicative repair. Together, these data suggest that CAF-I has a role in error-free postreplicative damage repair and may also have an auxiliary role in other repair mechanisms. Like the CAC genes, RAD6 is also required for gene silencing at telomeres. We find an increased loss of telomeric gene silencing in rad6Delta cac1Delta and rad18Delta cac1Delta double mutants, suggesting that CAF-I and multiple factors in the postreplicative repair pathway influence chromosome structure.

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dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.

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The yeast Saccharomyces provides a powerful system for investigating the biological effects of ionizing radiation, and studies have identified double-strand breaks in DNA as probably of critical importance in determining lethality. These breaks are normally repaired by a recombinational mechanism, but in the absence of repair a single break may be lethal to the cell. Genetic and molecular analysis of the eight genes known as RAD50 to RAD57 has revealed much about recombinational repair. These genes are also of central importance in other processes including meiosis and may be of general significance in understanding radiation responses in eukaryotes.

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Pulsed-field gel electrophoresis (PFGE) has been used to study the timing, frequency, and distribution of double-strand breaks (DSBs) in chromosomal-sized DNA during meiosis in yeast. It has previously been shown that DSBs are associated with some genetic hotspots during recombination, and it is important to know whether meiotic recombination events routinely initiate via DSBs. Two strains have been studied here--a high-sporulating homothallic wild type and a congenic mutant strain carrying a rad50S mutation. This mutant has previously been reported to accumulate broken molecules in meiosis to much higher frequencies than wild type and to abolish the characteristic wild-type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has been assessed primarily on Chromosome III and Chr. XV, using Southern hybridization to identify these chromosomes and their fragments. At any one time, break frequency in wild type is much lower than the cumulative frequency of recombination events that occur during meiosis. However, there is suggestive evidence that each break is short-lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of recombination events in wild type. However, since rad50S is substantially defective in completing recombination, it is not known for certain if it initiates events at wild-type frequencies. A surprising feature of the data is that a strong banding pattern is observed in the fragment distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur almost anywhere in the genome, although some regions are much hotter than others. Possible reasons for this apparent paradox are discussed. It may in part result from breakage levels too low for adequate detection in cold regions but may also imply that recombination events are localized more than previously realized. Alternatively, there may be a more indirect relationship between break sites and the associated recombination events.

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We describe a method for determining the position of a cloned internal sequence with respect to the ends of a DNA molecule. The molecules are randomly broken at low frequency and the fragments are subjected to electrophoresis. Southern hybridization using the cloned DNA as a probe identifies only those fragments containing the sequence. The size distribution of these fragments is such that two threshold changes in intensity of signal are seen in the smear pattern below the unbroken molecules. The positions of the changes represent the distances from the sequence to each molecular end. The intensity changes arise because the natural ends of the molecules influence the fragment distribution obtained. From once-broken molecules, no fragments can arise that contain a given sequence and are shorter than the distance between that sequence and the nearest molecular end. We tested the method by using x-rays to induce breakage in yeast DNA. Genes of independently known position were mapped within whole chromosomes or Not I restriction fragments using Southern blots from gels of irradiated molecules. We present equations to predict fragment distribution as a function of break-frequency and position of the probed sequence.

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We describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome III (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. We demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints, we present data on the timing of commitment to meiotic recombination scored genetically. We have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-sized circles originating in part from sister-chromatid exchange, which we find to be frequent during meiosis.

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The dihydrofolate reductase gene (DFR1) from Saccharomyces cerevisiae has been mapped and sequenced. The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim. A 1.8-kb SalI-BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E. coli DHFR-deficient (folA) mutant. Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted Mr of 24230. The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources. The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage. The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control. Several potential 'TATA' boxes are located in the sequence 5' to the gene. Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast. We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.

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The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways.

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The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.

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The effects of UV irradiation on DNA metabolism during meiosis have been examined in wild-type (RAD+) and mitotically defined excision-defective (rad1-1) strains of Saccharomyces cerevisiae that exhibit high levels of sporulation. The rad1-1 gene product is not required for normal meiosis: DNA synthesis, RNA synthesis, size of parental and newly synthesized DNA and sporulation are comparable in RAD+ and rad1-1 strains. Cells were UV irradiated at the beginning of meiosis, and the fate of UV-induced pyrimidine dimers as well as changes in DNA and DNA synthesis were followed during meiosis. Excision repair of pyrimidine dimers can occur during meiosis and the RAD1 gene product is required; alternate excision pathways do not exist. Although the rate of elongation is decreased, the presence of pyrimidine dimers during meiosis in the rad1-1 strain does not block meiotic DNA synthesis suggesting a bypass mechanism. The final size of DNA is about five times the distance between pyrimidine dimers after exposure to 4 J/m2. Since pyrimidine dimers induced in parental strands of rad1-1 prior to premeiotic DNA synthesis do not become associated with newly synthesized DNA, the mechanism for replicational bypass does not appear to involve a recombinational process. The absence of such association indicates that normal meiotic recombination is also suppressed by UV-induced damage in DNA; this result at the molecular level is supported by observations at the genetic level.

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The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD+) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m2 to the rad1 strain. Exposure to 25 J/m2 had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.

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The kinetic patterns of DNA synthesis in wild-type (RAD+) and rad 52 mutants of yeast, which exhibit high levels of synchrony during meiosis, are comparable. However, RAD 52 mutants accumulate single-strand breaks in parental DNA during the DNA synthesis period. Thus, the product of the RAD 52 gene has a role in meiotic DNA metabolism, as well as in the repair of DNA damage during mitotic growth. The observed breaks may be unresolved recombination intermediates.

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The biosynthesis of thymine nucleotides in Saccharomyces cerevisiae can be inhibited either by genetic lesions in the structural gene for thymidylate synthetase (TMP1) or by drugs that prevent the methylation of dUMP to dTMP. This methylation can be blocked by folate antagonists. We find that 5-fluoro-dUMP (FdUMP) is also an effective inhibitor in vivo. Inhibition of dTMP biosynthesis by these three different routes causes thymineless death. In addition to being cytotoxic, we find that FdUMP is highly recombinagenic in yeast but does not induce nuclear gene mutations. Provision of exogenous dTMP eliminates this induced mitotic recombination and cell killing. Similar results were obtained when a thymineless condition was provoked in cells by antifolate drugs or by dTMP deprivation in strains auxotrophic for this nucleotide. These findings show that, in contrast to the situation in prokaryotes, starvation for thymine nucleotides in yeast induces genetic recombination but is not mutagenic.

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In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.

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The temperature-sensitive Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase, and the DNA synthesized at the restrictive temperature contains many single-strand breaks. We find that holding a diploid homozygous for cdc9 at the restrictive temperature and then plating cells at the permissive temperature gives rise to increased intragenic and intergenic recombination. In the latter case, recombinants signaled by the ade2 locus rise to about 4% of the survivors after 6 hr of incubation at the restrictive temperature. We propose that the single-strand breaks left in DNA synthesized at the restrictive temperature may lead to recombination.

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Seven temperature-sensitive mutants have been isolated in Saccharomyces cerevisiae which show a reproducible defect in DNA synthesis at the restrictive temperature. One of these is allelic with rna11 (Hartwell et al., 1970) but the remaining mutants define six complementation groups and probably represent six different genes. The gene symbol dds (for depressed DNA synthesis) is proposed. At the restrictive temperature, rna11-2, dds2-1 and dds6-1 show a rapid and almost total cessation of DNA and RNA synthesis, whilst protein synthesis continues for several hours. The remaining dds mutants show a reduced rate of DNA synthesis from the time of temperature shift (dd1, dds3, dds4) or a cessation of DNA synthesis at a later time (dds5). In some cases, RNA synthesis is affected concomitantly with, or soon after, the depression in DNA synthesis. Possible reasons for the phenotypes of these mutants, and for the relative absence of yeast mutants which are unambiguously and specifically affected in DNA synthesis, are discussed. In addition, we report the isolation of seven new alleles of known cdc genes and ten new mutants with a cell cycle phenotype that complement those already known.

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Genetic tests with the yeast cell-cycle mutant cdc21 isolated by Hartwell indicate that the CDC21 gene in yeast is the same as the TMP1 gene, whose mutant alleles confer an auxotrophic requirement for thymidine-5'-monophosphate (dTMP). Yeast strains carrying cdc21 can grow at 37 degrees in the presence of dTMP provided that they are premeable to this compound. The gene is shown to be linked to ade2 on Chr. XV, and a case of intragenic complementation between cdc21 and another tmp1 allele is reported.

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