Cell Cycle Regulation of Mcm Localization

描述（由申请人提供）：我们正在使用芽殖酵母酿酒酵母（Saccharomyces cerevisiae）来了解真核细胞如何将DNA复制限制在每个细胞周期一轮。这种调节的破坏可能导致基因组不稳定，这是癌症的标志之一。防止再复制的一种保守机制是在复制起始发生后将起始I蛋白从细胞核中排除。在芽殖酵母中，异六聚体Mcm 2 -7复制复合物的核排斥通过细胞周期蛋白依赖性激酶Cdc 28调节Mcm核质转运而发生。我们已经确定了核定位信号（NLS）的Mcm 2和Mcm 3和一个潜在的核输出信号（内斯）的Mcm 3。我们还表明，Cdc 28磷酸化Mcm 3附近的NLS和内斯促进净核输出的Mcm 2 -7复合物。这种磷酸化可以通过抑制核输入、刺激核输出或两者兼而有之来发挥作用。为了研究这些可能性，我将追求以下具体目标： 目的1：确定Cdc 28是否以及如何调节Mcm核输入 目的2：确定Cdc 28是否以及如何调节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