Molecular Anal. of Pericentric Sister Chromatid Cohesion

描述（由申请人提供）：染色体的准确分离是遗传信息成功传递到子细胞的关键。这一过程中的缺陷与肿瘤发生、流产和先天性疾病有关。在有丝分裂过程中，复制的姐妹染色单体之间凝聚力的建立及其在中期的维持是姐妹染色单体分离的必要前提。在成对的姐妹染色单体上靠近动粒的凝聚力是特别强大的，并且被认为在空间上将动粒限制在促进双极微管附着形成的方向上。现在出现的证据表明，动粒也表现为一个增强剂的招聘凝聚素，凝聚蛋白的多亚基复合物，在大的臂间域。该提案的长期目标是使用酿酒酵母模型系统来阐明动粒协调微管结合和内聚活动的分子机制，以及这些活动如何发挥作用以维持基因组稳定性。我们的重点将是确定这些扩展的臂间粘连蛋白结构域的功能意义和在其产生的kinetochore-microtubule附件的作用。染色体分离的保真度将在缺失臂间粘连蛋白结构域后以及当用于粘连蛋白结合的着丝粒增强子的活性受限时进行遗传学检查。可以构建潜在的边界元件的缺失和插入，以确定这些元件是否有助于产生不同的臂间粘连蛋白结构域。标准技术可用于测试这些区域是否具有介导边界功能的特化染色质结构，并且将进行酵母单杂交筛选以鉴定边界活性所需的蛋白质因子。将进行高拷贝抑制筛选以鉴定将粘附素募集至臂间DNA的因子，并且将对在该筛选中鉴定的新蛋白进行标准遗传和生化分析以评估体内功能。着丝粒增强子通过着丝粒-微管相互作用的调节将在具有改变的微管结合活性的突变体中进行检查，并且微管附着的相对贡献和由这些附着对增强子活性调节产生的张力将使用仅能形成单极纺锤体微管附着的着丝粒来确定。

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