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）是防止非整倍体的机制之一，是基因组稳定性的重要调节因子。SAC是由着丝粒调控的，在本提案中，一个主要的未解决的问题是确定着丝粒如何测量张力和占用的缺乏，以及着丝粒如何将这些信息传递给细胞周期机制。我们将利用功能缺失突变体完成着丝点内SAC张力分支的高分辨率定位。我们已经开发了一种针对Mad3的磷酸化特异性抗体，这是SAC张力分支的第一个生化标记，我们建议使用它来解剖SAC调节的这一重要组成部分。我们提出了一个关于着丝点在张力检查点中的作用的新模型。我们将确定含有Mad3的蛋白亚群是否被磷酸化，Mad3在细胞中被磷酸化的位置，以及哪些着丝点蛋白需要传递张力信号。我们已经分离出了SAC占用分支特有的新型突变体。我们将分析SAC在DNA损伤途径中的作用，这需要SAC蛋白的Mec1和Tel1磷酸化，并代表有丝分裂调节的新途径。总之，本提案中的实验将为SAC信号如何启动和整合到细胞周期提供重要的见解。公共卫生相关性：染色体分离错误导致染色体数目不平衡（非整倍体），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防止由基因组不稳定引起的疾病的机制之一。本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.