Abstract
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, BER-defective cells and BER/NER-defective cells possess elevated levels of unrepaired, spontaneous oxidative DNA damage. This system allowed establishment of a range of approximately 400 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.
Highlights
Our results demonstrate that cells acquire remarkably high levels of spontaneous DNA damage that are normally repaired by base excision repair (BER) and nucleotide excision repair (NER)
Spontaneous Oxidative DNA Damage Accumulates When Excision Repair Is Compromised—To investigate the effects of unrepaired, spontaneous oxidative DNA damage on various biological and genetic endpoints, we first determined the relative levels of nuclear oxidative DNA damage in the wild type (WT), BERdefective, NER-defective, and BER/NER-defective strains via a gene-specific damage-detection assay
The BER/NER-defective large cell type ROS profile is intermediate to that produced by exposures to 3 and 4 mM H2O2 in the WT strain (Fig. 5B). These results indicate that the BER/NER-defective strain has adapted to survive under conditions of oxidative stress, which, when given to WT cells as an acute exposure to H2O2, produce similar intracellular ROS levels that are accompanied by a high degree of toxicity
Summary
Media, and Growth Conditions—All strains used in this study are isogenic derivatives of SJR751 (MAT␣ ade2–101oc his3⌬200 ura3⌬Nco lys2⌬Bgl leu2-R). Cells were treated with 2.5 mM H2O2 (Sigma) for 30 min at 30 °C and harvested by centrifugation and washed twice with sterile water. This DNA extraction procedure was designed to minimize the level of adventitious DNA damage. With an aliquot of this culture, fresh YPD media was inoculated to a density allowing harvest of ϳ2 ϫ 107 cells/ml after 15 h of growth. Following a 5-min centrifugation, 200 l of the hybridization mixture was loaded onto YSG98 microarray chips (Affymetrix, Santa Clara, CA) containing ϳ6400 ORFs.C. O. The chips were loaded into the fluidics station for washing and staining as described, Chapter 4 of the Affymetrix GeneChip Expression Analysis Technical Manual. Expression information can be found on the Doetsch laboratory website (www.biochem.emory.edu/labs/medpwd/microarray_index.html)
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