Biochemical Analysis of Sister Chromatid Cohesion

描述(申请人摘要)：姐妹染色单体凝聚力是必不可少的 确保基因信息忠实分离的细胞过程 在有丝分裂和减数分裂期间。这个项目的长期目标是了解 这一过程的生化基础是使用来自于 非洲爪哇的卵和哺乳动物组织培养细胞。一个 将重点放在结构和功能的表征上 粘附素，一种高度保守的蛋白质复合体，在 姐妹染色单体凝聚力的建立和维持。粘附素复合体是 由两个SMC(染色体结构维持)亚基和AT组成 至少两个非S MC亚基，预计将作为分子的一部分 将两个姐妹染色单体结合在一起的“胶”。在本建议中，(1) 将结合生物化学和结构方法来理解 粘附素复合体作用的分子机制。(2)细胞周期依赖性 粘附素和染色质之间的相互作用将在体外重建，并 装载和卸载过程的分子基础将被确定。(3) BimD和Scc2的脊椎动物同源物，这两种蛋白质在遗传上与 姐妹染色单体凝聚力，将在 无细胞萃取物和组织培养细胞中。(4)一种新的体外试验将 来确定姐妹染色单体凝聚力是如何在功能上偶联的 到DNA复制。从这项工作中获得的信息最终将 有助于更好地了解人类健康，因为染色体 异常，如非整倍体和易位，与 肿瘤的发展和出生缺陷。

DESCRIPTION (APPLICANT'S ABSTRACT): Sister chromatid cohesion is an essential cellular process that ensures the faithful segregation of genetic information during mitosis and meiosis. The long-term goal of this project is to understand the biochemical basis of this process by using cell-free extracts derived from Xenopus laevis (African toad) eggs and mammalian tissue culture cells. An emphasis will be made on the structural and functional characterization of cohesin, a highly conserved protein complex that plays a key role in establishing and maintaining sister chromatid cohesion. The cohesin complex is composed of two SMC (structural maintenance of chromosomes) subunits and at least two non-S MC subunits, and is predicted to act as part of the molecular "glue" that holds two sister chromatids together. In this proposal, (1) Biochemical and structural approaches will be combined to understand the molecular mechanisms of action of the cohesin complex. (2) Cell cycle-dependent interactions between cohesin and chromatin will be reconstituted in vitro and the molecular basis of loading and unloading processes will be determined. (3) Vertebrate homologs of BimD and Scc2, two proteins implicated genetically in sister chromatid cohesion, will be identified and characterized in the cell-free extracts and in tissue culture cells. (4) A novel in vitro assay will be developed to determine how sister chromatid cohesion is functionally coupled to DNA replication. The information obtained from this work will ultimately contribute to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development and birth defects.