Molecular Anal. of Pericentric Sister Chromatid Cohesion

分子肛门。

基本信息

批准号：
6905508
负责人：
PAUL Connor MEGEE
金额：
$ 25.92万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-01 至 2007-06-30
项目状态：
已结题

项目摘要

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

描述（由申请人提供）：染色体的准确分离是将遗传信息成功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。复制的姐妹染色单体之间的内聚力的建立及其在中期的维持是有丝分裂期间姐妹染色单体分离的必要先决条件。配对姐妹染色单体上靠近动粒的内聚力特别强，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着丝粒还充当在大的中心周区域招募粘连蛋白（粘连蛋白的多亚基复合物）的增强子。该提案的长期目标是利用酿酒酵母模型系统来阐明动粒协调微管结合和内聚活性的分子机制，以及这些活性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中心周粘连蛋白结构域的功能意义以及动粒-微管附着在其生成中的作用。在删除中心周粘连蛋白结构域后以及当着丝粒增强子对粘连蛋白结合的活性受到限制时，将通过遗传学方法检查染色体分离的保真度。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周中心粘连域。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殊染色质结构，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，以确定将粘连蛋白募集到中心周DNA的因子，并且在该筛选中鉴定的新蛋白质将进行标准遗传和生化分析，以评估体内功能。将在具有改变的微管结合活性的突变体中检查着丝粒增强子通过着丝粒-微管相互作用的调节，并且将使用可以确定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产生的张力来确定形成单极纺锤体微管附件