Environmental pollutants and carcinogens may cause genetic damage either by acting as direct mutagens or by disrupting the checkpoints that regulate the cell cycle and maintain genetic stability. Ran1 is the prototype of a novel class of small, Ras-like GTPases members of which play essential roles in the control of DNA replication and chromosome condensation/decondensation. Recent studies in the fission yeast S. pombe, suggest that partial loss of Ran function can cause genetic instability and aneuploidy. A similar loss may account for the aneuploidy frequently found in human tumors. Despite the importance of this class of small GTPase, however, it remains unknown how many members of the Ran family exist, what the functions of Ran and its homologs are, at the molecular level, how Ran GTPase activity is regulated, what factors Ran proteins interact with in the nucleus, and what role Ran may play in the mitotic checkpoints of the cell cycle. The purpose of this grant application is to answer these questions. cDNA libraries are being screened to identify Ran homologs. Antisera will be produced to GST-fusions of Ran homologs, and epitope-tagged versions of the proteins will be created. Subcellular localization and tissue distribution of the homologs will be determined. The hypothesis will be tested that Ran GTP/GDP ratios are regulated in a cell cycle-dependent fashion, and that this ratio changes either at The G1/S or the G2/M boundaries. A second hypothesis to be tested is that Ran proteins are components of cell cycle check points, and that disruption of Ran function can cause genetic instability. This hypothesis will be tested by micro- injection of Ran mutants, and by measuring the frequency of CAD gene amplification following treatment of Ran-transfected cells with the cytotoxic drug, PALA. Finally, to begin to elucidate the molecular mechanisms by which Ran exerts its effects on the cell cycle, proteins that interact specifically with Ran will be identified and cloned, by use of GST-fusion affinity matrices, and by use of the yeast two hybrid system. Overall, these studies will yield information on the functions and regulation of important GTP-binding proteins which play essential roles in the control of the cell cycle. The studies will also provide information on potential targets for xenobiotic toxins and carcinogens that induce genetic damage through mechanisms other than direct mutagenesis.
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