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成功将遗传信息传播给子细胞的关键。此过程中的缺陷与肿瘤发生，流产和先天性疾病有关。在有丝分裂过程中隔离姐妹染色单体的基本先决条件是复制的姐妹染色单体与其通过中期维持之间的凝聚力的建立。在配对姐妹染色单体上的动力学上的凝聚力尤其强大，被认为可以在空间上限制动力学的方向，从而促进了促进双极微管附着的形成。现在有证据表明，动力学也是大型周围域上凝聚蛋白的凝聚蛋白（一种多元素络合物络合物）的募集的增强剂。该提案的长期目标是使用酿酒酵母模型系统来阐明动物学辅助辅助微管结合和凝聚活性的分子机制，以及这些活动如何保持基因组稳定性。我们的重点是确定这些扩展的周围粘着蛋白结构域的功能意义以及动型微管附着在其一代中的作用。在缺失上百年粘着蛋白结构域以及centromeric增强剂的粘合素结合的活性时，将对染色体隔离的保真度进行遗传检查。构建潜在边界元素WiFi的删除和插入，以确定这些元素是否有助于创建不同的周期粘着蛋白结构域。标准技术WIFI用于测试这些区域是否具有介导边界功能的专门染色质结构，并将执行酵母单杂交筛网以识别边界活动所需的蛋白质因子。将进行高拷贝抑制筛查，以确定将粘质募集到周围的DNA的因素，并在此筛选中鉴定出的新型蛋白质将经过标准的遗传和生化分析，以评估体内功能。在具有改变微管结合活性改变的突变体中，将检查通过动力学 - 微管相互作用对中心粒增强剂的调节，以及微管附件的相对贡献以及这些附件对增强剂活性调节所产生的张力，可以使用单粒子固定的中心固定链球链球固定链球固定链球固定链球固定。