Regulation of chromosome segregation

项目描述（申请人提供）：该项目将探索控制染色体分离起始的调控系统，染色体分离起始是细胞生命中的关键事件，也是肿瘤发生过程中经常出错的事件。在细胞周期的S期染色体复制之后，产生的姐妹染色单体通过一种称为内聚蛋白的蛋白质复合体连接在一起。在有丝分裂过程中，姐妹染色单体对在双极性有丝分裂纺锤体上定向。在中期和后期转变中，姐妹染色单体之间的内聚蛋白连接突然被一种称为分离酶的蛋白酶溶解，导致姐妹染色单体同步分离并向纺锤体的相反极移动。提出的研究将探索芽殖酵母中姐妹染色单体分离的控制，其中我们对这一过程的许多知识是首次发现的。这项工作的一个关键目标将是确定和表征产生非常强大的调控机制，后期调控系统的开关行为。在对两条染色体上携带荧光标记的酵母细胞的初步研究中，发现姐妹染色单体分离的同步性部分依赖于控制分离酶激活的正反馈回路。这些研究还发现，染色体IV始终先于染色体V分离，这表明染色体是按照特定的顺序分离的。提出的研究的第一个目的将是进一步表征酵母中多染色体分离的同步性和顺序，并解决不同染色体有序分离的一般机制。第二个目标将是重建从纯化组分中分离姐妹染色单体的生化步骤，允许在体外详细研究分离酶激活和内聚蛋白裂解。最后，第三个目标将是使用这些细胞和生化工具来解决控制黏结蛋白分离酶活性的机制，重点是通过控制黏结蛋白磷酸化的蛋白激酶和磷酸酶来调节黏结蛋白裂解。从这些研究中获得的知识将为染色体分离的控制提供新的见解-染色体分离的错误通常会导致发育问题和癌症进展。

DESCRIPTION (provided by applicant): The project will explore the regulatory system that controls the initiation of chromosome separation, a critical event in the life of the cell and an event that often goes awry during tumorigenesis. Following duplication of the chromosomes in S phase of the cell cycle, the resulting sister chromatids are linked together by a protein complex called cohesin. During mitosis, the sister-chromatid pairs are oriented on the bipolar mitotic spindle. At the metaphase-anaphase transition, the cohesin linkage between sister's chromatids is abruptly dissolved by a protease called separase, resulting in synchronous separation of sister chromatids and their movement to opposite poles of the spindle. The proposed studies will explore the control of sister-chromatid separation in the budding yeast Saccharomyces cerevisiae, where much of our knowledge of this process was first uncovered. A key goal of the work will be to identify and characterize the regulatory mechanisms that generate the remarkably robust, switch-like behavior of the anaphase regulatory system. In preliminary studies with yeast cells carrying fluorescent tags on two chromosomes, the synchrony of sister-chromatid separation was found to depend in part on a positive feedback loop that governs activation of separase. These studies also led to the discovery that Chromosome IV consistently separates before Chromosome V, suggesting that chromosomes separate in a specific sequence. The first aim of the proposed studies will be to further characterize synchrony and order in the separation of multiple chromosomes in yeast, and to address the general mechanisms underlying the ordered separation of different chromosomes. The second aim will be to reconstitute the biochemical steps of sister-chromatid separation from purified components, allowing detailed studies of separase activation and cohesin cleavage in vitro. Finally, the third aim will be to use these cellular and biochemical tools to address the mechanisms governing separase activity toward cohesin, with an emphasis on the regulation of cohesin cleavage by protein kinases and phosphatases that control cohesin phosphorylation. The knowledge gained from these studies will provide new insights into the control of chromosome segregation - errors in which often contribute to developmental problems and cancer progression. PUBLIC HEALTH RELEVANCE: When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in this process can result in genetic damage or defects in chromosome number, which can accelerate cancer progression or cause developmental defects. The proposed studies focus on the regulatory system that controls the initiation of chromosome separation, with an emphasis on the mechanisms underlying the remarkable robustness and accuracy of this system. These studies will lead to a better understanding of how errors in chromosome segregation can arise in human disease.