title: RAD53 is limiting in double-strand break repair and in protection against toxicity associated with ribonucleotide reductase inhibition.
abstract: The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G₂ cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.
authors.affiliation: Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
authors.forename: Shay
authors.initials: S
authors.lastname: Covo
authors.forename: James W
authors.initials: JW
authors.lastname: Westmoreland
authors.forename: Amit K
authors.initials: AK
authors.lastname: Reddy
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Gordenin
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D019610:Cytoprotection / Q000187:drug effects / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004351:Drug Resistance / Q000187:drug effects / Q000528:radiation effects; D042822:Genomic Instability / Q000187:drug effects / Q000528:radiation effects; D006918:Hydroxyurea / Q000633:toxicity*; D050296:Microbial Viability / Q000187:drug effects / Q000528:radiation effects; D009153:Mutagens / Q000633:toxicity; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects / Q000528:radiation effects
chemical_list: D018797:Cell Cycle Proteins; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D012264:Ribonucleotide Reductases; C551024:Rnr1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea