Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae

In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine. We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase. These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.     

Structure of DNA polymerase delta from Saccharomyces cerevisiae

DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations. Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P. M., and Gerik, K. J. (1998) J. Biol. Chem. 273, 19756-19762). We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta. The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm). Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.     

DNA polymerase zeta introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae

Spontaneous DNA damage can be dealt with by multiple repair/bypass pathways that have overlapping specificities. We have used a frameshift reversion assay to examine spontaneous mutations that accumulate in yeast strains defective for the high-fidelity nucleotide excision repair or recombination pathways. In contrast to the simple frameshift mutations that occur in wild-type strains, the reversion events in mutant strains are often complex in nature, with the selected frameshift mutation being accompanied by one or more base substitutions. Genetic analyses demonstrate that the complex events are dependent on the Pol zeta translesion polymerase, thus implicating the DNA damage bypass activity of low-fidelity translesion polymerases in hypermutation phenomena.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase delta

DNA polymerase delta (pol delta) is a high fidelity eukaryotic enzyme that participates in DNA repair and is essential for DNA replication. Toward the goal of dissecting its multiple biological functions, here we describe the biochemical properties of Saccharomyces cerevisiae pol delta with a methionine replacing conserved leucine 612 at the polymerase active site. Compared with wild type pol delta, L612M pol delta has normal processivity and slightly higher polymerase specific activity. L612M pol delta also has normal 3' exonuclease activity, yet it is impaired in partitioning mismatches to the exonuclease active site, thereby reducing DNA synthesis fidelity. Error rates in vitro for L612M pol delta are elevated for both base substitutions and single base deletions but in a highly biased manner. For each of the six possible pairs of reciprocal mismatches that could arise during replication of complementary DNA strands to account for any particular base substitution in vivo (e.g. T-dGMP or A-dCMP for T to C transitions), L612M pol delta error rates are substantially higher for one mismatch than the other. These results provide a biochemical explanation for our observation, which confirms earlier genetic studies, that a haploid pol3-L612M S. cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo.     

Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions

Eukaryotic DNA polymerase delta (Pol delta) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion of individual dNTPs opposite a template guanine, Pol delta is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3'-exonuclease activity, implies that Pol delta should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol delta does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations/100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol delta confirm the high nucleotide selectivity of the polymerase and further indicate that proofreading enhances the base substitution fidelity of the wild type enzyme by at least 60-fold. However, Pol delta inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions/100,000 nucleotides polymerized. Moreover, wild type Pol delta frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol delta and Pol epsilon both have high base substitution fidelity, Pol delta is much less accurate than Pol epsilon for deletions involving repetitive sequences. Thus, strand slippage during replication by wild type Pol delta may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.     

Suppressors of the temperature sensitivity of DNA polymerase α mutations in Saccharomyces cerevisiae

We have isolated two high copy, allele-specific suppressors of the temperature sensitivity of mutations in POL1, the gene that encodes the catalytic subunit of DNA polymerase α in the yeast Saccharomyces cerevisiae. Both genes, PSP1 and PSP2, also partially suppressed a mutation in POL3 which encodes DNA polymerase δ, and both also affected a mutation in CDC6, which acts in initiation of DNA replication. Suppression was not general, since ts mutations in several genes unrelated to replication were not affected. PSP1 was partially effective on low-copy-number vectors, while PSP2 required high copy numbers. The presence of suppressing plasmids did not alter the steady-state level of Pol1 protein, so suppression does not appear to be due to an increase in production or stability of Pol1p. Deletion of either PSP gene or both in combination resulted in apparently normal viable cells. While neither gene is homologous to genes with known functions, PSP1 and PSP2 both have unusual amino acid compositions: PSP1 is rich in asparagine and glutamine, while PSP2 is rich in asparagine and contains “RGG” motifs that have been associated with RNA-binding proteins. We also describe a transposon-mediated strategy that should be generally effective for rapid characterization of multicopy suppressors.     

Epistatic interactions of spontaneous mutations in haploid strains of the yeast Saccharomyces cerevisiae

Several important biological phenomena, including genetic recombination and sexual reproduction, could have evolved to counteract genome contamination by deleterious mutations. This postulate would be especially relevant if it were shown that deleterious mutations interact in such a way that their individual negative effects are reinforced by each other. The hypothesis of synergism can be tested experimentally by crossing organisms bearing deleterious mutations and comparing the fitness of the parents and their progeny. The present study used laboratory strains of the budding yeast burdened with mutations resulting from absence of a major DNA mismatch repair function. Only in one, or possibly two, crosses out of eight did fitness of the progeny deviate from that of their parents in a direction indicating synergism. Furthermore, the distributions of progeny fitness were not skewed as would be expected if strong interactions were present. The choice of experimental material ensured that genetic recombination was extensive, all four meiotic products were available for fitness assays, and that the mutations were probably numerous. Despite this generally favourable experimental setting, synergism did not appear to be a dominating force shaping fitness of yeast containing randomly generated mutations.     

Mitochondrial mutations restricting spontaneous translational frameshift suppression in the yeast Saccharomyces cerevisiae.

Summary The +1 frameshift mutation, M5631, which is located in the gene (oxi1) for cytochrome c oxidase II (COXII) of the yeast mitochondrial genome, is suppressed spontaneously to a remarkably high extent (20%–30%). The full-length wild-type COXII produced as a result of suppression allows the mutant strain to grow with a leaky phenotype on non-fermentable medium. In order to elucidate the factors and interactions involved in this translational suppression, the strain with the frameshift mutation was mutated by MnCl₂ treatment and a large number of mutants showing restriction of the suppression were isolated. Of 20 mutants exhibiting a strong, restricted, respiration-deficient (RD) phenotype, 6 were identified as having mutations in the mitochondrial genome. Furthermore, genetic analyses mapped one mutation to the vicinity of the gene for tRNA^Pro and two others to a region of the tRNA cluster where two-thirds of all mitochondrial tRNA genes are encoded. The degree of restriction of the spontaneous frameshift suppression was characterized at the translational level by in vivo ³⁵S-labeling of the mitochondrial translational products and immunoblotting. These results showed that in some of these mutant strains the frameshift suppression product is synthesized to the same extent as in the leaky parent strain. It is suggested that more than one +1 frame-shifted product is made as a result of suppression in these strains: one is as functional as the wild-type COXII, the other(s) is (are) non-functional and prevent leaky growth on non-fermentable medium. A possible mechanism for this heterogenous frameshift suppression is discussed.     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.     

A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase delta, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.     

Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.     

Nuclear mutations in Saccharomyces cerevisiae which increase the spontaneous mutation frequency in mitochondrial DNA.

Summary Fourteen mutants have been identified in which the frequency of spontaneous mutations in mitochondrial DNA is increased. As well as increasing the frequency of mutations to resistance to erythromycin, oligomycin and spiramycin, all the mutants also show changes in the frequency of spontaneous petite induction. None of the mutants has any effect on the frequency of spontaneous nuclear mutations. Nine of the mutants are in one complementation group and five are in another. The phenotype of both groups is caused by a single nuclear mutation.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit- mutant of Saccharomyces cerevisiae

In cultures of the mit- mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit- cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a 32P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the ori1 sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

Hierarchical and metabolic regulation of glucose influx in starved Saccharomyces cerevisiae

A novel method dissecting the regulation of a cellular function into direct metabolic regulation and hierarchical (e.g., gene-expression) regulation is applied to yeast starved for nitrogen or carbon. Upon nitrogen starvation glucose influx is down-regulated hierarchically. Upon carbon starvation it is down-regulated both metabolically and hierarchically. The method is expounded in terms of its implications for diverse types of regulation. It is also fine-tuned for cases where isoenzymes catalyze the flux through a single metabolic step.     

Estimates of the rate and distribution of fitness effects of spontaneous mutation in Saccharomyces cerevisiae

The per-genome, per-generation rate of spontaneous mutation affecting fitness (U) and the mean fitness cost per mutation (s) are important parameters in evolutionary genetics, but have been estimated for few species. We estimated U and sh (the heterozygous effect of mutations) for two diploid yeast strains differing only in the DNA mismatch-repair deficiency used to elevate the mutation rate in one (mutator) strain. Mutations were allowed to accumulate in 50 replicate lines of each strain, during 36 transfers of randomly chosen single colonies (approximately 600 generations). Among wild-type lines, fitnesses were bimodal, with one mode showing no change in mean fitness. The other mode showed a mean 29.6% fitness decline and the petite phenotype, usually caused by partial deletion of the mitochondrial genome. Excluding petites, maximum-likelihood estimates adjusted for the effect of selection were U = 9.5 x 10(-5) and sh = 0.217 for the wild type. Among the mutator lines, the best fit was obtained with 0.005 < or = U < or = 0.94 and 0.049 > or = sh > or = 0.0003. Like other recently tested model organisms, wild-type yeast have low mutation rates, with high mean fitness costs per mutation. Inactivation of mismatch repair increases the frequency of slightly deleterious mutations by approximately two orders of magnitude.