Mitotic regulation by checkpoints in budding yeast

摘要 染色体分离的错误是非整倍体的主要来源，也是染色体非整倍体形成的驱动力。 肿瘤的发展。主轴装配检查点（SAC）是 防止非整倍性，是基因组稳定性的重要调节剂。战略空军司令部精心策划 一个主要的未解决的问题，在这个建议中解决，是确定 动粒如何测量缺乏紧张和缺乏占有， 动粒将信息传递给细胞周期机制。我们将完成高 使用丢失的分辨率映射动粒内SAC的张力分支， 功能突变体我们已经开发了一种磷酸化特异性抗体Mad3，这是第一个 这是SAC张力分支的生化标志物，我们建议用它来解剖这个分支。 是SAC监管的重要组成部分。我们提出了一个新的模式， 紧张检查点的动粒。我们将确定是否含有Mad3的蛋白质的子集 是磷酸化的，其中Mad3在细胞中被磷酸化， 来传递张力信号我们分离出了新的突变体， SAC的占用分支。我们将剖析SAC在DNA损伤中的作用， 该途径需要SAC蛋白的Mec1和Tel 1磷酸化，代表了一种新的 有丝分裂调控途径。总之，本提案中的实验将提供重要的 深入了解SAC信号是如何启动和整合到细胞周期中的。

Abstract Errors in chromosome segregation are the major source of aneuploidy and a driving force in the development of tumors. The spindle assembly checkpoint (SAC) is one of the mechanisms that prevents aneuploidy and is an important regulator of genomic stability. The SAC is orchestrated by the kinetochore and a major unresolved problem, addressed in this proposal, is to determine how the kinetochore measures the lack of tension and lack of occupancy and how the kinetochore transfers that information to the cell cycle machinery. We will complete high resolution mapping of the tension branch of the SAC within the kinetochore using loss-of- function mutants. We have developed a phospho-specific antibody to Mad3 that is the first biochemical marker for the tension branch of the SAC and we propose to use it to dissect this important component of SAC regulation. We propose a new model for the role of the kinetochore in the tension checkpoint. We will determine if subsets of Mad3-containing proteins are phosphorylated, where Mad3 is phosphorylated in the cell and which kinetochore proteins are needed to transmit the tension signal. We have isolated novel mutants that are specific to the occupancy branch of the SAC. We will dissect the role for the SAC in the DNA damage pathway which requires Mec1 and Tel1 phosphorylation of SAC proteins and represents a novel pathway of mitotic regulation. Together, the experiments in this proposal will provide important insights into how SAC signaling is initiated and integrated into the cell cycle.