This proposal centers on the cellular response to DNA damage. The experiments are performed in the fission yeast Schizosaccharomyces pombe, a model system that has provided a paradigm of cell cycle and checkpoint control. S. pombe has two major checkpoint responses that monitor genome integrity. These are the intra-S-phase checkpoint and the DNA damage checkpoint. The intra-S-phase checkpoint is activated when the progression of DNA replication is impeded, and though its effector kinase Cds1, the stability of stalled replication forks is maintained to facilitate resumption of DNA replication. All forms of DNA damage activate the DNA damage checkpoint, and through its effector kinase Chk1, the cell cycle is halted such that the lesion can be repaired prior to entry into mitosis. These checkpoints are key determinants of genome integrity, and as such their function is crucial to prevent the genome instability that is a hallmark of cancer. Checkpoints are also crucial in the biology of cellular ageing and senescence, and in the response environmental mutagens. Thus, a detailed understanding of their biology is central to human disease. There are outstanding issues as to how these checkpoints are coordinated, how they are initiated and how they are terminated. It is the long-term goal of our laboratory, and of this project, to identify all components of these checkpoints and fill holes in the current understanding of the checkpoint signaling pathways. We present a significant body of preliminary data in which novel aspects of checkpoint signaling have been uncovered. First, we have begun to determine mechanisms of activation and inactivation of Chk1, an important issue in both basic biology and in the development of anti-cancer drugs that target Chk1. Second, we identify new regulatory mechanisms through which cells prevent DNA damage during blocks to DNA replication. These are via novel checkpoint-mediated regulation of Topoisomerase I molecules at the replication forks. Further, we have identified two additional new checkpoint genes that are implicated in the initiating events of checkpoint signaling. Both genes, when overexpressed, restore function to a hypomorphic allele of chk1, and appear to be amplifying the initiating checkpoint signal. Null alleles of these genes show they are required for the DNA damage checkpoint. They appear to function at the most upstream end of the checkpoint pathway, which include the least understood aspects of checkpoint control. To further these preliminary studies, we propose three aims utilizing genetic, biochemical and cell biological approaches. First, we dissect mechanisms of Chk1 activation and inactivation using a unique series of reagents we have developed for the S. pombe enzyme. Second, we dissect Topoisomerase I mediated checkpoint activation, and how this enzyme is regulated by the intra-S- phase checkpoint. Finally, we characterize a family of related nucleases that appear to be processing primary lesions into checkpoint activating single-stranded DNA. Given the high conservation of checkpoint gene function, these new checkpoint components are likely to function similarly in human cells.
Cells proliferate with remarkable fidelity, and defects in quality controls, known as checkpoints, are at the root cause of cancer, aging and responses to environmental mutagens. Because these processes are ancient in origin, we use simple yeast cells to identify new genes that are relevant to human disease. Our proposal analyses three new genes in the response of cells to DNA damage.
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