DEREGULATING CYCLIN DEPENDENT KINASE

细胞周期蛋白依赖性激酶（Cdk's）在所有真核生物的细胞周期调控中都是至关重要的。Cdk受到细胞周期蛋白结合的严格调节，因此细胞周期蛋白的可用性是调节细胞周期进程的主要手段之一。这一提议是为了检验放松这一制度的后果。在一组实验中，提出了一种独立于细胞周期蛋白结合要求的出芽酵母Cdk Cdc28的突变激活策略。实现这一目标（已经取得了重大进展）将允许解剖细胞周期蛋白在简单酶激活中的作用，而不是将酶靶向特定底物。在第二组实验中，我们建议检查使特定b型细胞周期蛋白/Cdc28复合物Clb5-Cdc28的水平和活性在细胞周期中构成的后果。尽管目前的模型预测，由于DNA复制起源的功能受到干扰，这应该会使细胞周期停止在G1期，但我们的研究结果表明，情况可能并非如此。我们建议通过细胞周期来描述Clb5降解的控制，以及Clb5破坏盒在其降解中的作用。我们提出实验来验证Clb5本质上专门用于进入S期，而另一种b型细胞周期蛋白Clb2本质上专门用于进入有丝分裂的假设。这一假设与目前所有b型细胞周期蛋白对DNA复制的正调控和负调控能力相同的模型相反。

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.