In most eukaryotic cells the decision to enter into the cell division cycle is made during the G1 phase. A recent confluence of many different lines of research in a variety of organisms has shown that the transient accumulation of active G1 cyclin/cyclin-dependent kinase (Cdk) complexes triggers commitment to DNA replication and cell division during G1 phase. This discovery, in turn, has nurtured the unifying hypothesis that the intracellular and extracellular signals that either encourage or restrain cell division do so by modulating the concentration of active G1 cyclin/Cdk complexes. Cell cycle control in the yeast Saccharomyces cerevisiae is achieved with components that are structurally and functionally homologous to those that reign in vertebrate cells. A large group of CDC genes that promote different stages in the cell division cycle pathway have been identified by the isolation of yeast mutants that fail to divide. Among these genes are G1 cyclins and a Cdk required for progression from G1 to S phase. Dr. Deshaies has devised an in vitro system that recapitulates two key aspects of the dynamic regulation of Gl cyclin/Cdk complexes. Cyclin-depleted and G1-arrested yeast extract supplemented with recombinant G1 cyclin sustains the complex molecular choreography that yields active Cdk protein kinase, and subsequently leads to the extensive ligation of ubiquitin to cyclin. This in vitro system provides a unique opportunity to link the advantage of molecular genetic analysis in yeast with the incisive resolution afforded by biochemical reconstitution. Research described in this application proposes to exploit the complementary benefits of genetics and biochemistry to (i) investigate the biochemical pathway that specifies formation of active G1 cyclin/Cdk complexes, (ii) assess the role of the CDC37 gene in the formation of active G1 cyclin/Cdk complexes, (iii) investigate the biochemical pathway that triggers the ubiquitination of G1 cyclin by the Cdc34p ubiquitin-conjugating enzyme, and (iv) test the role of ubiquitin-mediated cyclin destruction in normal cell cycle control. Recent results suggest that defects in regulatory proteins that modulate G1 cyclin/Cdk activity may unleash cancer in up to half of its victims. Currently, there exists only a sketchy molecular roadmap for Cdk regulation and G1 progression in human cells. The functional conservation of cell cycle control proteins from yeast to man, however, provides great hope that studies in tractable model organisms such as yeast will advance our understanding of G1 control in human cells. Delineation of the biochemical reactions that govern the formation and destruction of active G1 cyclin/Cdk complexes in vitro will provide insight into how these processes are deployed in normal and diseased cells, and may assist the design or discovery of agents with propitious effects in neoplastic cells.
