Molecular Anal. of Pericentric Sister Chromatid Cohesion

描述(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。这一过程中的缺陷与肿瘤形成、流产和先天性疾病有关。复制的姐妹染色单体之间凝聚力的建立和中期保持这种凝聚力是姐妹染色单体在有丝分裂过程中分离的必要条件。在配对的姐妹染色单体上，动点附近的凝聚力特别强，被认为空间地限制了动点的取向，促进了双极微管附着的形成。现在出现的证据表明，着丝粒也作为增强剂在大的着丝粒周围区域招募粘附素，粘附素是一种凝聚蛋白的多亚单位复合体。这一建议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凝聚活动的分子机制，以及这些活动如何发挥作用以维持基因组的稳定。我们的重点将是确定这些延伸的着丝粒粘连蛋白结构域的功能意义以及动粒-微管连接在它们发生中的作用。染色体分离的保真度将在着丝粒周围粘连蛋白结构域缺失和着丝粒增强子对粘连蛋白结合的活性受到限制后进行遗传学检查。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着丝粒周围粘连蛋白结构域。标准技术将被用来测试这些区域是否具有调节边界功能的特殊染色质结构，并将进行酵母单杂交筛选以确定边界活动所需的蛋白质因子。将进行高拷贝抑制物筛选，以确定将粘附素招募到着丝粒周围DNA的因素，并对在此筛选中确定的新蛋白进行标准的遗传和生化分析，以评估体内功能。动粒-微管相互作用对着丝粒增强子的调节作用将在微管结合活性改变的突变体中进行检验，微管附着的相对贡献以及这些附着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