Cell Cycle Regulation of Mcm Localization

描述(由申请人提供)：我们正在使用萌芽酵母酿酒酵母来了解真核细胞如何将DNA复制限制在每个细胞周期一轮。这一调控的中断可能导致基因组不稳定，这是癌症的特征之一。防止再次复制的一种保守机制是在复制起始发生后将起始I蛋白排除在细胞核之外。在萌芽酵母中，异六聚体Mcm2-7复制复合体的核排斥是通过细胞周期蛋白依赖的激酶CDC28调节MCM的核质运输而发生的。我们在McM2和MCM3上发现了核定位信号(NLSS)，在MCM3上发现了潜在的核输出信号(NES)。我们还表明，MCM3在其NLS和NES附近的CDC28磷酸化促进了McM2-7复合体的净核输出。这种磷酸化可以通过抑制核进口、刺激核出口或两者兼而有之来发挥作用。为了调查这些可能性，我将追求以下具体目标： 目标1：确定CDC28是否以及如何管制MCM核进口 目标2：确定CDC28是否以及如何管制MCM核出口

DESCRIPTION (provided by applicant): We are using the budding yeast Saccharomyces cerevisiae to understand how eukaryotic cells limit DNA replication to one round per cell cycle. Disruption of this regulation can lead to genomic instability, one of the hallmarks of cancer. One conserved mechanism to prevent re-replication is to exclude initiation I proteins from the nucleus after replication initiation has occurred. In budding yeast, nuclear exclusion of the heterohexameric Mcm2-7 replication complex occurs through regulation of Mcm nucleocytoplasmic transport by the cyclin-dependent kinase Cdc28. We have identified nuclear localization signals (NLSs) on Mcm2 and Mcm3 and a potential nuclear export signal (NES) on Mcm3. We have also shown that Cdc28 phosphorylation of Mcm3 near its NLS and NES promotes the net nuclear export of the Mcm2-7 complex. This phosphorylation could work by inhibiting nuclear import, stimulating nuclear export, or doing both. To investigate these possibilities, I will pursue the following specific aims: Aim 1: Determine whether and how Cdc28 regulates Mcm nuclear import Aim 2: Determine whether and how Cdc28 regulates Mcm nuclear export