Molecular Anal. of Pericentric Sister Chromatid Cohesion

描述（由申请人提供）：染色体的准确分离是成功将遗传信息传递给子细胞的关键。这一过程的缺陷与肿瘤发生、流产和先天性疾病有关。姐妹染色单体间内聚的建立和维持是姐妹染色单体在有丝分裂过程中分离的必要前提。姐妹染色单体上靠近着丝点的内聚力特别强，并且被认为在空间上约束着丝点在促进双极微管附着物形成的方向。现在有证据表明，着丝点也可以作为内聚蛋白（一种多亚基复合物）在大的周中心结构域上募集的增强剂。本研究的长期目标是利用酿酒酵母模型系统阐明着丝点协调微管结合和内聚活动的分子机制，以及这些活动如何维持基因组的稳定性。我们的重点将是确定这些扩展的中心周围内聚蛋白结构域的功能意义以及着丝点-微管附着物在其产生中的作用。染色体分离的保真度将在基因上进行检查，在删除一个中心周围的粘聚蛋白结构域和当着丝粒增强子的粘聚蛋白结合的活性是有限的。可以构建潜在边界元素的删除和插入，以确定这些元素是否有助于创建不同的中心周围内聚域。标准技术将用于测试这些区域是否具有介导边界功能的特殊染色质结构，酵母单杂交筛选将用于鉴定边界活性所需的蛋白质因子。将进行高复制抑制筛选，以确定将内聚蛋白招募到中心周围DNA的因素，并在该筛选中发现的新蛋白质将进行标准的遗传和生化分析，以评估体内功能。着丝点-微管相互作用对着丝粒增强子的调节将在微管结合活性改变的突变体中进行研究，微管附着物的相对贡献以及这些附着物对增强子活性调节的张力将使用只能形成单极纺锤体微管附着物的着丝粒来确定

DESCRIPTION (provided by applicant): The accurate segregation of chromosomes is key to the successful transmission of genetic information to daughter cells. Deficiencies in this process are associated with tumorigenesis, miscarriages, and congenital disorders. The establishment of cohesion between replicated sister chromatids and its maintenance through metaphase are essential prerequisites for the segregation of sister chromatids during mitosis. Cohesion proximal to the kinetochores on paired sister chromatids is especially robust, and is thought to sterically constrain the kinetochores in an orientation that promotes the formation of bipolar microtubule attachments. Evidence is now emerging that the kinetochore also behaves as an enhancer for the recruitment of cohesin, a multisubunit complex of cohesion proteins, over large pericentric domains. The long-term goal of this proposal is to use the Saccharomyces cerevisiae model system to elucidate the molecular mechanism through which the kinetochore coordinates both microtubule binding and cohesion activities and how these activities function to maintain genomic stability. Our focus will be to determine the functional significance of these extended pericentric cohesin domains and the role of kinetochore-microtubule attachments in their generation. The fidelity of chromosome segregation will be examined genetically following the deletion of a pericentric cohesin domain and when the activity of the centromeric enhancer for cohesin binding is limited. Deletions and insertions of potential boundary elements wifi be constructed to determine whether these elements contribute to the creation of distinct pericentric cohesin domains. Standard techniques wifi be used to test whether these regions possess specialized chromatin structures that mediate boundary function, and a yeast one-hybrid screen will be performed to identify protein factors required for boundary activity. A high copy suppressor screen will be performed to identify factors that recruit cohesin to pericentric DNA and novel proteins identified in this screen will be subjected to standard genetic and biochemical analyses to assess in vivo function. The regulation of the centromeric enhancer by kinetochore-microtubule interactions will be examined in mutants with altered microtubule binding activities and the relative contributions of microtubule attachments and the tension that results from these attachments to the regulation of enhancer activity will be determined using a centromere that can form oniy a monopolar spindle microtubule attachment