Mitotic regulation by checkpoints in budding yeast

描述（由申请人提供）：染色体分离错误是非整倍体的主要来源，也是肿瘤发展的驱动力。纺锤体组装检查点（SAC）是防止非整倍性的机制之一，是基因组稳定性的重要调节因子。SAC是由动粒精心策划的，并且在本提案中解决的一个主要未解决的问题是确定动粒如何测量缺乏张力和缺乏占用以及动粒如何将该信息传递给细胞周期机器。我们将使用功能丧失突变体完成动粒内SAC张力分支的高分辨率映射。我们已经开发了一种磷酸化特异性抗体Mad 3，这是第一个生化标志物的紧张分支的SAC，我们建议使用它来剖析这一重要组成部分的SAC调节。我们提出了一个新的模型中的紧张检查点的动粒的作用。我们将确定是否Mad 3-含有蛋白质的子集被磷酸化，Mad 3在细胞中被磷酸化，以及需要哪些动粒蛋白来传递张力信号。我们已经分离出特异于SAC占据分支的新突变体。我们将剖析SAC在DNA损伤途径中的作用，该途径需要SAC蛋白的Mec 1和Tel 1磷酸化，并代表了一种新的有丝分裂调控途径。总之，本提案中的实验将为SAC信号传导如何启动并整合到细胞周期中提供重要的见解。公共卫生相关性：染色体分离的错误导致染色体数量的不平衡（非整倍性），这是某些出生缺陷的标志，也是癌症发展的主要驱动力。纺锤体组装检查点是防止非整倍体和预防基因组不稳定性引起疾病的机制之一。该提案中的实验将为纺锤体检查点如何组织，如何调节以及如何整合到细胞周期中提供重要见解。长期目标是通过预防导致出生缺陷和癌症的非整倍体来改善人类健康。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Errors in chromosome segregation result in imbalances in chromosome number (aneuploidy), a hallmark of some kinds of birth defects and a major driving force in the development of cancer. The spindle assembly checkpoint is one of the mechanisms that prevents aneuploidy and guards against the diseases resulting from genomic instability. The experiments in this proposal will provide important insights into how the spindle checkpoint is organized, how it is regulated and how it is integrated into the cell cycle. The long term goal is to improve human health by preventing aneuploidy that results in birth defects and cancer.