Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

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.     

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.     

Interplay of DNA repair pathways controls methylation damage toxicity in Saccharomyces cerevisiae

Methylating agents of S(N)1 type are widely used in cancer chemotherapy, but their mode of action is poorly understood. In particular, it is unclear how the primary cytotoxic lesion, O(6)-methylguanine ((Me)G), causes cell death. One hypothesis stipulates that binding of mismatch repair (MMR) proteins to (Me)G/T mispairs arising during DNA replication triggers cell-cycle arrest and cell death. An alternative hypothesis posits that (Me)G cytotoxicity is linked to futile processing of (Me)G-containing base pairs by the MMR system. In this study, we provide compelling genetic evidence in support of the latter hypothesis. Treatment of 4644 deletion mutants of Saccharomyces cerevisiae with the prototypic S(N)1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) identified MMR as the only pathway that sensitizes cells to MNNG. In contrast, homologous recombination (HR), postreplicative repair, DNA helicases, and chromatin maintenance factors protect yeast cells against the cytotoxicity of this chemical. Notably, DNA damage signaling proteins played a protective rather than sensitizing role in the MNNG response. Taken together, this evidence demonstrates that (Me)G-containing lesions in yeast must be processed to be cytotoxic.     

DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.     

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.     

Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae

Oxidative DNA damage is likely to be involved in the etiology of cancer and is thought to accelerate tumorigenesis via increased mutation rates. However, the majority of malignant cells acquire a specific type of genomic instability characterized by large-scale genomic rearrangements, referred to as chromosomal instability (CIN). The molecular mechanisms underlying CIN are not entirely understood. We utilized Saccharomyces cerevisiae as a model system to delineate the relationship between genotoxic stress and CIN. It was found that elevated levels of chronic, unrepaired oxidative DNA damage caused chromosomal aberrations at remarkably high frequencies under both selective and nonselective growth conditions. In this system, exceeding the cellular capacity to appropriately manage oxidative DNA damage resulted in a “gain-of-CIN” phenotype and led to profound karyotypic instability. These results illustrate a novel mechanism for genome destabilization that is likely to be relevant to human carcinogenesis.     

DNA polymerase 4 of Saccharomyces cerevisiae is important for accurate repair of methyl-methanesulfonate-induced DNA damage

The DNA polymerase 4 protein (Pol4) of Saccharomyces cerevisiae is a member of the X family of DNA polymerases whose closest human relative appears to be DNA polymerase lambda. Results from previous genetic studies conflict over the role of Pol4 in vivo. Here we show that deletion of Pol4 in a diploid strain of the SK1 genetic background results in sensitivity to methyl methanesulfonate (MMS). However, deletion of Pol4 in other strain backgrounds and in haploid strains does not yield an observable phenotype. The MMS sensitivity of a Pol4-deficient strain can be rescued by deletion of YKu70. We also show that deletion of Pol4 results in a 6- to 14-fold increase in the MMS-induced mutation frequency and in a significant increase in AT-to-TA transversions. Our studies suggest that Pol4 is critical for accurate repair of DNA lesions induced by MMS.     

Differential repair of UV damage in Saccharomyces cerevisiae.

Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed.     

Efficient repair of large DNA loops in Saccharomyces cerevisiae

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.     

Kinetochores prevent repair of UV damage in Saccharomyces cerevisiae centromeres

Centromeres form specialized chromatin structures termed kinetochores which are required for accurate segregation of chromosomes. DNA lesions might disrupt protein-DNA interactions, thereby compromising segregation and genome stability. We show that yeast centromeres are heavily resistant to removal of UV-induced DNA lesions by two different repair systems, photolyase and nucleotide excision repair. Repair resistance persists in G(1)- and G(2)/M-arrested cells. Efficient repair was obtained only by disruption of the kinetochore structure in a ndc10-1 mutant, but not in cse4-1 and cbf1 Delta mutants. Moreover, UV photofootprinting and DNA repair footprinting showed that centromere proteins cover about 120 bp of the centromere elements CDEII and CDEIII, including 20 bp of flanking CDEIII. Thus, DNA lesions do not appear to disrupt protein-DNA interactions in the centromere. Maintaining a stable kinetochore structure seems to be more important for the cell than immediate removal of DNA lesions. It is conceivable that centromeres are repaired by postreplication repair pathways.     

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na2SeO3), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.     

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.     

Saccharomyces cerevisiae DNA repair processes: an update

The effect of incubating T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly , and isoforms) was assessed by immunoblotting, enzyme activity assay and [³H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC and PKC had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca²⁺) indicated that translocation of PKC and was very rapid — detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight (authentic) PKC immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC moved more rapidly than PKC which appeared to translocate more quickly than PKC . After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC was not translocated by LHRH. (Mol Cell Biochem 165: 65–75, 1996)