Genetic alterations leading to increases in internal potassium concentrations are detrimental for DNA integrity in Saccharomyces cerevisiae (original) (raw)
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Molecular and cellular biology, 2008
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 rele...
Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae
Molecular and cellular biology, 1996
DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were ...
Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast
Cell cycle (Georgetown, Tex.), 2015
DNA damage tolerance (DDT) mechanisms allow cells to synthesize a new DNA strand when the template is damaged. Many mutations resulting from DNA damage in eukaryotes are generated during DDT when cells use the mutagenic translesion polymerases, Rev1 and Polζ, rather than mechanisms with higher fidelity. The coordination among DDT mechanisms is not well understood. We used live-cell imaging to study the function of DDT mechanisms throughout the cell cycle of the fission yeast Schizosaccharomyces pombe. We report that checkpoint-dependent mitotic delay provides a cellular mechanism to ensure the completion of high fidelity DDT, largely by homology-directed repair (HDR). DDT by mutagenic polymerases is suppressed during the checkpoint delay by a mechanism dependent on Rad51 recombinase. When cells pass the G2/M checkpoint and can no longer delay mitosis, they completely lose the capacity for HDR and simultaneously exhibit a requirement for Rev1 and Polζ. Thus, DDT is coordinated with t...
Checkpoint Control of DNA Repair in Yeast
2021
Budding yeast has been a model organism for understanding how DNA damage is repaired and how cells minimize genetic instability caused by arresting or delaying the cell cycle at well-defined checkpoints. However, many DNA damage insults are tolerated by mechanisms that can both be error-prone and error-free. The mechanisms that tolerate DNA damage and promote cell division are less well-understood. This review summarizes current information known about the checkpoint response to agents that elicit both the G2/M checkpoint and the intra-S phase checkpoint and how cells adapt to unrepaired DNA damage. Tolerance to particular bulky DNA adducts and radiomimetic agents are discussed, as well as possible mechanisms that may control phosphatases that deactivate phosphorylated proteins.
The Ppz protein phosphatases regulate Trk-independent potassium influx in yeast
2004
The Ppz protein phosphatases have been recently shown to negatively regulate the major potassium transport system in the yeast Saccharomyces cerevisiae, encoded by the TRK1 and TRK2 genes. We have found that, in the absence of the Trk system, Ppz mutants require abnormally high concentrations of potassium to proliferate. This can be explained by the observation that trk1 trk2 ppz1 or trk1 trk2 ppz1 ppz2 strains display a very poor rubidium uptake, with markedly increased K m values. These cells are very sensitive to the presence of several toxic cations in the medium, such as hygromicyn B or spermine, but not to lithium or sodium cations. At limiting potassium concentrations, addition of EGTA to the medium improves growth of these mutants. Therefore, our results indicate that, in addition to their role in regulating Trk potassium transporters, Ppz phosphatases (essentially Ppz1) positively affect the residual low affinity potassium transport mechanisms in yeast. These findings may provide a new way to elucidate the molecular nature of the low affinity potassium uptake system in yeast as well as a useful model to analyze the function of plant or mammalian potassium channels through heterologous expression in yeast.
Journal of Biological Chemistry, 2002
We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5-DNA kinase and 3-DNA phosphatase activities in vitro. Furthermore, we detected 3-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1⌬ strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to ␥-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1⌬ cells restores resistance to ␥-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.
DNA damage bypass pathways and their effect on mutagenesis in yeast
FEMS Microbiology Reviews, 2020
ABSTRACTWhat is the origin of mutations? In contrast to the naïve notion that mutations are unfortunate accidents, genetic research in microorganisms has demonstrated that most mutations are created by genetically encoded error-prone repair mechanisms. However, error-free repair pathways also exist, and it is still unclear how cells decide when to use one repair method or the other. Here, we summarize what is known about the DNA damage tolerance mechanisms (also known as post-replication repair) for perhaps the best-studied organism, the yeast Saccharomyces cerevisiae. We describe the latest research, which has established the existence of at least two error-free and two error-prone inter-related mechanisms of damage tolerance that compete for the handling of spontaneous DNA damage. We explore what is known about the induction of mutations by DNA damage. We point to potential paradoxes and to open questions that still remain unanswered.
Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments
Genes to Cells, 2002
Background: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions. Results: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase δ δ δ δ. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells. Conclusions: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.
Journal of Biological Chemistry, 2004
To determine the spectrum of effects elicited by specific levels of spontaneous DNA damage, a series of isogenic Saccharomyces cerevisiae strains defective in base excision repair (BER) and nucleotide excision repair (NER) were analyzed. In log phase of growth, when compared with wild type (WT) or NER-defective cells, BERdefective cells and BER/NER-defective cells possess elevated levels of unrepaired, spontaneous oxidative DNA damage. This system allowed establishment of a range of ϳ400 to 1400 Ntg1p-recognized DNA lesions per genome necessary to provoke profound biological changes similar in many respects to the phenotypic properties of cancer cells. The BER/NER-defective cells are genetically unstable, exhibiting mutator and hyper-recombinogenic phenotypes. They also exhibit aberrations in morphology, DNA content, and growth characteristics compared with WT, BER-defective, and NER-defective cells. The BER/NER-defective cells also possess increased levels of intracellular reactive oxygen species, activate the yeast checkpoint response pathway via Rad53p phosphorylation in stationary phase, and show profound changes in transcription patterns, a subset of which can be ascribed to responses resulting from unrepaired DNA damage. By establishing a relationship between specific levels of spontaneous DNA damage and the ensuing deleterious biological consequences, these yeast DNA excision repair-defective strains are an informative model for gauging the progressive biological consequences of spontaneous DNA damage accumulation and may have relevancy for delineating underlying mechanisms in tumorigenesis.