Cyclin-dependent kinases (Cdk's) are critical in regulation of the cell cycle in all eukaryotes. Cdk's are stringently regulated by cyclin binding, and cyclin availability thus is one of the main means by which cell cycle progression is regulated. This proposal is for experiments to examine the consequences of deregulation of this system. In one set of experiments, a strategy is proposed for mutational activation of the budding yeast Cdk Cdc28 independent of the cyclin binding requirement. Achievement of this goal (towards which significant progress has been made) will allow dissection of the roles of cyclins in simple enzyme activation as opposed to targeting of the enzyme to specific substrates. In a second set of experiments, we propose examining the consequences of making the level and activity of a particular B-type cyclin/Cdc28 complex, Clb5-Cdc28, constitutive through the cell cycle. Although current models predict that this should halt the cell cycle in G1 phase due to interference with function of origins of DNA replication, our results suggest that this may not be the case. We propose to characterize control of Clb5 degradation through the cell cycle, and the role of the Clb5 destruction box in its degradation. We propose experiments to test the hypothesis that Clb5 is intrinsically specialized for S phase entry while another B-type cyclin, Clb2, is intrinsically specialized for entry into mitosis. This hypothesis is in contrast to current models in which all B-type cyclins are equally capable of both positive and negative regulation of DNA replication.
| Cross, F R; Levine, K (2000) Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase. Genetics 154:1549-59 |
| Levine, K; Kiang, L; Jacobson, M D et al. (1999) Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase. Mol Cell 4:353-63 |
