CELL CYCLE CHECKPOINT CONTROL IN RESPONSE TO DNA DAMAGE

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The long-term goal of this project is to determine how cells regulate progression through the cell cycle when their DNA has been damaged. It has long been known that eukaryotic cells will delay progression through the cell cycle when the integrity of the genome has been compromised. In the last decade it has become clear that genetically defined signal transduction pathways, known as cell cycle checkpoints, couple the detection of DNA damage to control of cell cycle progression. The replication of damaged DNA templates or the segregation of damaged chromosomes can have catastrophic consequences for the integrity of the genome. Therefore, these checkpoint mechanisms play essential roles in maintaining genomic stability and, when compromised, can contribute to the onset of cancer or to cell death. Mutations in the human checkpoint pathway gene ATM, lead to the genetic disorder Ataxia Telangiectasia which is characterized by progressive neurodegeneration and a high incidence of cancer. The fission yeast, Schizosaccharomyces pombe, has proven to be an outstanding model system for identifying components of the cell cycle regulatory machinery as well as the DNA damage checkpoint pathway. The protein kinase Chk1, first identified in fission yeast, is required for cell cycle arrest when DNA is damaged. Homologues of Chkl have been identified in a variety of eukaryotic organisms including frogs, flies, worms and humans. In addition, an & pombe homologue of the ATM gene, rad3, has been shown to function on the same pathway as Chk1. To understand the role played by Chk1 in the DNA damage checkpoint pathway, several approaches will be taken that capitalize on the ease with which genetic and biochemical analyses can be carried out with & pombe. Proteins that interact with Chk1 or which influence the activity of Chk1 will be identified in genetic screens that make use of novel checkpoint defective alleles of the chkl gene. The previously identified interaction between Chk1 and Rad24, another protein shown to play a role on the DNA damage checkpoint pathway, will be characterized in detail. Determinants of the subcellular localization of Chkl will be identified as the location of Chk1 in the cell has important implications both for the nature of signaling to Chk1 as well as for the nature of putative Chk1 targets. The requirement of posttranslational modification of Chk1 by phosphorylation will be analyzed by identifying the sites of phosphorylation induced by DNA damage and analyzing the importance of those sites for Chkl function