Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Citation - WoS: 19Citation - Scopus: 22Absence of Superoxide Dismutase Activity Causes Nuclear Dna Fragmentation During the Aging Process(Academic Press Inc., 2014) Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koç, AhmetSuperoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.Article Citation - WoS: 12Citation - Scopus: 14Roles of Atr1 Paralogs Ymr279c and Yor378w in Boron Stress Tolerance(Elsevier Ltd., 2011) Bozdağ, Gönensin Ozan; Uluışık, İrem; Gülcüler, Gülce Sıla; Karakaya, Hüseyin Çağlar; Koç, AhmetBoron is a necessary nutrient for plants and animals, however excess of it causes toxicity. Previously, Atr1 and Arabidopsis Bor1 homolog were identified as the boron efflux pump in yeast, which lower the cytosolic boron concentration and help cells to survive in the presence of toxic amount of boron. In this study, we analyzed ATR1 paralogs, YMR279c and YOR378w, to understand whether they participate in boron stress tolerance in yeast. Even though these genes share homology with ATR1, neither their deletion rendered cells boron sensitive nor their expression was significantly upregulated by boron treatment. However, expression of YMR279, but not YOR378w, from the constitutive GAPDH promoter on a high copy plasmid provided remarkable boron resistance by decreasing intracellular boron levels. Thus our results suggest the presence of a third boron exporter, YMR279c, which functions similar to ATR1 and provides boron resistance in yeast.Article Citation - WoS: 18Citation - Scopus: 19Assessment of Chronological Lifespan Dependent Molecular Damages in Yeast Lacking Mitochondrial Antioxidant Genes(Elsevier Ltd., 2010) Demir, Ayşe Banu; Koç, AhmetThe free radical theory of aging states that oxidative damage to biomolecules causes aging and that antioxidants neutralize free radicals and thus decelerate aging. Mitochondria produce most of the reactive oxygen species, but at the same time have many antioxidant enzymes providing protection from these oxidants. Expecting that cells without mitochondrial antioxidant genes would accumulate higher levels of oxidative damage and, therefore, will have a shorter lifespan, we analyzed oxidative damages to biomolecules in young and chronologically aged mutants lacking the mitochondrial antioxidant genes: G. RX2, CCP1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, and PRX1. Among these mutants, ccp1Δ, trx3Δ, grx5Δ, prx1Δ, mutants were sensitive to diamide, and ccs1Δ and sod2Δ were sensitive to both diamide and menadione. Most of the mutants were less viable in stationary phase. Chronologically aged cells produced higher amount of superoxide radical and accumulated higher levels of oxidative damages. Even though our results support the findings that old cells harbor higher amount of molecular damages, no significant difference was observed between wild type and mutant cells in terms of their damage content. © 2010 Elsevier Inc.Article Citation - WoS: 30Citation - Scopus: 32Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast(American Chemical Society, 2010) Kaya, Alaattin; Koç, Ahmet; Lee, Byung Cheon; Fomenko, Dmitri E.; Rederstorff, Mathieu; Krol, Alain; Lescure, Alain; Gladyshev, Vadim N.Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments. © 2010 American Chemical Society.Article Citation - WoS: 55Citation - Scopus: 64Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast(American Society for Microbiology, 2009) Kaya, Alaattin; Karakaya, Hüseyin Çağlar; Fomenko, Dmitri E.; Gladyshev, Vadim N.; Koç, AhmetBoron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1△ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1△ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1△ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Article Citation - WoS: 17Citation - Scopus: 19Effect of Thioredoxin Deletion and P53 Cysteine Replacement on Human P53 Activity in Wild-Type and Thioredoxin Reductase Null Yeast(American Chemical Society, 2009) Stoner, Christopher S.; Pearson, George D.; Koç, Ahmet; Merwin, Jason R.; Lopez, Nathan I.; Merrill, Gary FredericReporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (K d = 0.9 μM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Δtrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.Article Citation - WoS: 7Citation - Scopus: 8Checkpoint Deficient Rad53-11 Yeast Cannot Accumulate Dntps in Response To Dna Damage(Elsevier Ltd., 2007) Koç, Ahmet; Merrill, Gary F.Deoxyribonucleotide pools are maintained at levels that support efficient and yet accurate DNA replication and repair. Rad53 is part of a protein kinase regulatory cascade that, conceptually, promotes dNTP accumulation in four ways: (1) it activates the transcription of ribonucleotide reductase subunits by inhibiting the Crt1 repressor; (2) it plays a role in relocalization of ribonucleotide reductase subunits RNR2 and RNR4 from nucleus to cytoplasm; (3) it antagonizes the action of Sml1, a protein that binds and inhibits ribonucleotide reductase; and (4) it blocks cell-cycle progression in response to DNA damage, thus preventing dNTP consumption through replication forks. Although several lines of evidence support the above modes of Rad53 action, an effect of a rad53 mutation on dNTP levels has not been directly demonstrated. In fact, in a previous study, a rad53-11 mutation did not result in lower dNTP levels in asynchronous cells or in synchronized cells that entered the S-phase in the presence of the RNR inhibitor hydroxyurea. These anomalies prompted us to investigate whether the rad53-11 mutation affected dNTP levels in cells exposed to a DNA-damaging dose of ethylmethyl sulfonate (EMS). Although dNTP levels increased by 2- to 3-fold in EMS treated wild-type cells, rad53-11 cells showed no such change. Thus, the results indicate that Rad53 checkpoint function is not required for dNTP pool maintenance in normally growing cells, but is required for dNTP pool expansion in cells exposed to DNA-damaging agents.