The chronological life span of Saccharomyces cerevisiae

Simple model systems have played an important role in the discovery of fundamental mechanisms of aging. Studies in yeast, worms and fruit flies have resulted in the identification of proteins and signalling pathways that regulate stress resistance and longevity. New findings indicate that these pathways may have evolved to prevent damage and postpone aging during periods of starvation and may be conserved from yeast to mammals. We will review the yeast S. cerevisiae model system with emphasis on the chronological life span as a model system to study aging and the regulation of stress resistance in eukaryotes.     

Plasmid accumulation reduces life span in Saccharomyces cerevisiae

Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.     

Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae

Photoactivated psoralens used in treatment of skin diseases like Psoriasis and Vitiligo cause DNA damage, the repair of which may lead to mutations and thus to higher risk to have skin cancer. The simple eukaryote Saccharomyces cerevisiae was chosen to investigate the cells' genetic endowment with repair mechanisms for this type of DNA damage and to study the genetic consequences of such repair. Genetic studies on yeast mutants sensitive to photoactivated psoralens, named pso mutants, showed their allocation to 10 distinct loci. Cloning and molecular characterization allowed their grouping into three functional classes: (I) the largest group comprises seven PSO genes that are either generally or specifically involved in error-prone DNA repair and thus affect induced mutability and recombination; (II) one PSO gene that represents error-free excision repair, and (III) two PSO genes encoding proteins not influencing DNA repair but physiological processes unrelated to nucleic acid metabolism. Of the seven DNA repair genes involved in induced mutagenesis three PSO loci [PSO1/REV3, PSO8/RAD6, PSO9/MEC3] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of interstrand cross-link (ICL) that are produced in DNA by a variety of bi- and polyfunctional mutagens and that appears to be important for a likewise repair function in humans as well. In silico analysis predicts a putative endonucleolytic activity for Pso2p/Snm1p in removing hairpins generated as repair intermediates. The absence of induced mutation in pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation of translesion polymerase zeta in error-prone repair. Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity. The seventh gene, PSO10 which controls an unknown step in induced mutagenesis is not yet cloned. Two genes, PSO6/ERG3 and PSO7/COX11, are responsible for structural elements of the membrane and for a functional respiratory chain (RC), respectively, and their function thus indirectly influences sensitivity to photoactivated psoralens.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

DNA interstrand cross-link repair in Saccharomyces cerevisiae

DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.     

Effect of stress on the life span of the yeast Saccharomyces cerevisiae

A correlation is known to exist in yeast and other organisms between the cellular resistance to stress and the life span. The aim of this study was to examine whether stress treatment does affect the generative life span of yeast cells. Both heat shock (38 degrees C, 30 min) and osmotic stress (0.3 M NaCl, 1 h) applied cyclically were found to increase the mean and maximum life span of Saccharomyces cerevisiae. Both effects were more pronounced in superoxide dismutase-deficient yeast strains (up to 50% prolongation of mean life span and up to 30% prolongation of maximum life span) than in their wild-type counterparts. These data point to the importance of the antioxidant barrier in the stress-induced prolongation of yeast life span.     

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae.

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.     

Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases eta and zeta and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.     

Loss of heterozygosity and DNA damage repair in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) of tumor suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Understanding how LOH events arise may provide an opportunity for the prevention or early intervention of cancer development. In an effort to investigate the source of LOH events, we constructed MATalphacan1Delta::LEU2 and MATa CAN1 haploid yeast strains and examined canavanine-resistance mutations in a MATa CAN1/MATalphacan1Delta::LEU2 heterozygote formed by mating UV-irradiated and nonirradiated haploids. An increase in LOH was observed when the irradiated CAN1 haploid was mated with nonirradiated can1Delta::LEU2, while reversed irradiation only marginally increased LOH. In the rad51Delta background, allelic crossover type LOH increased following UV irradiation but not gene conversion. In the rad52Delta background, neither type of LOH increased. The chromosome structure following LOH and the requirement for Rad51 and Rad52 proteins indicated the involvement of gene conversion, allelic crossover and break-induced replication. We argued that LOH events could have occurred during the repair of double-strand breaks on a functional (damaged) but not nonfunctional (undamaged) chromosome through recombination.