the long-term objective of the proposed research is to elucidate the mechanisms that regulate the exit from mitosis in the fission yeast Schizosaccharomyces pombe. The isolation and characterization of the fission yeast dcd1-1ts mutant has led to the identification of two evolutionarily conserved proteins that are required for the complex structural changes associated with the completion of mitosis and re- establishment of the interphase state (Sazer and Nurse, 1992. The dcd1 gene encodes a protein similar to the human chromatin binding protein RCC1. A suppressor of dcd1-1ts, called fyt1, is nearly identical to a human GTP-binding protein in the family typified by the human proto- oncogene p21ras. The similarity of these two S. pombe proteins to human proteins of known function, and in the case of p21ras of known structure as well, provide a framework within which to design experiments examining the role of these and related proteins in mitotic exit. The knowledge gained from the proposed research should further our understanding of the role these human proteins play in cell cycle control, signal transduction pathways, and ras-mediated cancer. The dcd1 and fyt1 proteins are the core of a molecular switch that plays a key role in chromosome decondensation (Sazer & Nurse, 1992). the proposed research is aimed at understanding what turns this switch on and off and what happens when the switch is flipped. The four Specific Aims of the proposed research are to: A) Characterize cell cycle stage specific changes in the behavior of dcd1 and fyt1 proteins. Antibodies to these two proteins will be used to monitor cell cycle changes in their intracellular distribution and their association with the chromatin and each other; B) Identify the proteins that convey the signal for the completion of mitosis to dcd1. Genetic screens will be used to isolate both extragenic suppressors of dcd1-1ts and additional mutants with the dcd1-1ts phenotype; C) Identify proteins that are the effectors of fyt1 and/or the activators of fyt1 GTPase activity. Mutations will be introduced into the fyt1 gene in regions comparable to those in p21ras that are known to be important for effector interaction and for nucleotide binding and hydrolysis. Aberrant phenotypes of cells expressing these genes will be characterized and extragenic suppressors isolated; D) Isolate additional mutants defective in the ability to re-enter the cell cycle following mitosis. The pilot screen in which the dcd1-1ts mutant was identified will be continued to isolate new mutants defective in the mitosis-to-interphase transition.
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