Regulation of chromosome segregation

描述(由申请者提供)：该项目将探索控制染色体分离启动的调控系统，这是细胞生命中的一个关键事件，也是在肿瘤形成过程中经常出错的事件。在细胞周期的S期染色体复制后，产生的姐妹染色单体通过一种名为粘附素的蛋白质复合体连接在一起。在有丝分裂过程中，姐妹染色单体对定位在两极有丝分裂纺锤体上。在中期到后期的转变中，姐妹染色单体之间的粘附素连接被一种称为分离酶的蛋白酶突然分解，导致姐妹染色单体同步分离并移动到纺锤体的相反两极。拟议的研究将探索在萌芽酵母中姐妹染色单体分离的控制，我们对这一过程的许多知识最初是在酿酒酵母中发现的。这项工作的一个关键目标将是识别和表征产生后期调控系统非常健壮、开关样行为的调控机制。在对在两条染色体上携带荧光标记的酵母细胞进行的初步研究中，发现姐妹染色单体分离的同步性在一定程度上取决于控制分离酶激活的正反馈回路。这些研究还发现，第四染色体总是在第五染色体之前分离，这表明染色体在特定的序列中分离。拟议研究的第一个目标将是进一步表征酵母中多条染色体分离的同步性和有序性，并解决不同染色体有序分离的一般机制。第二个目标是重建从纯化成分中分离姐妹染色单体的生化步骤，以便在体外详细研究分离酶的激活和粘附素的切割。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分离酶对粘附素活性的机制，重点是通过控制粘附素磷酸化的蛋白激酶和磷酸酶来调节粘附素的切割。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--染色体分离中的错误往往会导致发育问题和癌症进展。 与公共卫生相关：当一个细胞繁殖时，染色体首先被复制，然后被分离成一对子细胞。这一过程中的错误可能会导致遗传损伤或染色体数量缺陷，从而加速癌症进展或导致发育缺陷。建议的研究集中在控制染色体分离启动的调控系统，重点是该系统显著的稳健性和准确性背后的机制。这些研究将有助于更好地理解人类疾病中染色体分离错误是如何发生的。

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.