Checkpoint kinase phosphorylation in response to endogenous oxidative DNA damage in repair-deficient stationary-phase Saccharomyces cerevisiae |
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Authors: | Vaibhav Pawar Liu Jingjing Nila Patel Nimrat Kaur Paul W. Doetsch Gerald S. Shadel Hong Zhang Wolfram Siede |
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Affiliation: | a Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, United States b Departments of Biochemistry, Radiation Oncology, and Hematology and Medical Oncology, and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, United States c Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States d Department of Cell Biology, Capital Medical University, Beijing 100069, PR China |
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Abstract: | Stationary-phase Saccharomyces cerevisiae can serve as a model for post-mitotic cells of higher eukaryotes. Phosphorylation and activation of the checkpoint kinase Rad53 was observed after more than 2 days of culture if two major pathways of oxidative DNA damage repair, base excision repair (BER) and nucleotide excision repair (NER), are inactive. The wild type showed a low degree of Rad53 phosphorylation when the incubation period was drastically increased. In the ber ner strain, Rad53 phosphorylation can be abolished by inclusion of antioxidants or exclusion of oxygen. Furthermore, this modification and enhanced mutagenesis in extended stationary phase were absent in rho° strains, lacking detectable mitochondrial DNA. This checkpoint response is therefore thought to be dependent on reactive oxygen species originating from mitochondrial respiration. There was no evidence for progressive overall telomere shortening during stationary-phase incubation. Since Rad50 (of the MRN complex) and Mec1 (the homolog of ATR) were absolutely required for the observed checkpoint response, we assume that resected random double-strand breaks are the critical lesion. Single-strand resection may be accelerated by unrepaired oxidative base damage in the vicinity of a double-strand break. |
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Keywords: | Yeast Stationary phase Oxidative DNA damage Checkpoints Aging |
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