The role of Cdc27 in miotic regulation of the APC/C

描述（由申请人提供）：染色体分离是每个生物体共同的最关键的过程之一。为了确保适当的分离，单元采用检查点机制来监视分离机制中的缺陷。有丝分裂检查点确保即使是单个不正确连接的染色体也能够延迟中期到后期的转变。为了实现染色体分离之前的延迟，检查点通过一种尚不清楚的机制抑制后期促进复合体/环体（APC/C）。我们的初步研究表明，注射针对APC/C亚基Cdc27的C端抗体，可以消除HeLa细胞中的有丝分裂检查点。在酵母中进行的初步研究表明，Cdc27的c端缺失在nocodazole的存在下会过早地分离其姐妹染色单体。为了扩展这些观察，我建议在出芽酵母酿酒酵母和哺乳动物组织培养系统中使用遗传和生化方法来表征Cdc27和有丝分裂检查点之间的相互作用，并测试这种相互作用如何促进APC/C的有丝分裂检查点抑制。

DESCRIPTION (provided by applicant): Chromosome segregation is one of the most critical processes common to every organism. To ensure proper segregation, the cell employs a checkpoint mechanism to monitor defects in the segregation machinery. The mitotic checkpoint ensures that even a single improperly attached chromosome is capable of delaying the metaphase to anaphase transition. To achieve a delay prior to chromosome segregation, the checkpoint inhibits, through a poorly understood mechanism, the anaphase-promoting complex/cyclosome (APC/C). Our preliminary studies revealed that injection of antibodies to the c-terminus of Cdc27, a subunit of the APC/C, abrogated the mitotic checkpoint in HeLa cells. This is supported by preliminary studies in yeast which revealed a c-terminal deletion of Cdc27 that prematurely separates its sister chromatids in the presence of nocodazole. To extend these observations, I propose to use both genetic and biochemical approaches in the budding yeast Saccharomyces cerevisiae and in a mammalian tissue culture system to characterize the interaction between Cdc27 and the mitotic checkpoint and test how this interaction might facilitate mitotic checkpoint inhibition of the APC/C.