Mechanisms of sister chromatid pairing

描述（由申请方提供）：细胞必须识别从S期到后期开始的染色体复制产物，称为姐妹染色单体。然而，姐妹染色单体第一次配对的机制仍然未知。CTF 7/ECO 1是凝聚力建立途径的创始成员，似乎将姐妹染色单体配对反应与DNA复制偶联。姐妹配对也可以在S期外诱导，表明Ctf 7 p建立活性受到严格调控。目前，Ctf 7 p仍然是已知的唯一必需的建立因子。最近的研究结果证实，Ctf 7 p的功能在S期，在S期被招募到染色质，并结合到许多染色质相关的复制因子。在第1阶段，我们将使用定点突变分析结合生化方法，以确定Ctf 7 p染色质募集所需的蛋白质相互作用以及Ctf 7 p募集/激活如何调节。然后将遗传和分子分析与高分辨率显微镜凝聚力测定配对，以测试新等位基因在细胞活力和姐妹配对反应中的作用。在第2阶段，我们将确定Ctf 7 p如何配对姐妹染色单体，一旦招募到染色质。我们将从我们的实验室中寻找Ctf 7 p与Pds 5 p相关的结果，Pds 5 p是一种维持姐妹配对所需的粘附素调节剂。Ctf 7 p-Pds 5 p结合是建立和维持内聚的过程之间唯一已知的联系。为了解决这些问题，我们将使用生物化学和细胞周期作图策略来测试Ctf 7 p-Pds 5 p结合是否是细胞周期特异性的、直接的和必需的。Ctf 7 p和Pds 5 p都是后修饰的。分子和生物化学方法将用于确定Ctf 7 p-Pds 5 p建立活性如何在细胞周期中受到调节、调节结合以及最终Ctf 7 p-Pds 5 p如何影响染色质上的粘着蛋白动力学。

DESCRIPTION (provided by applicant): Cells must identify the products of chromosome replication, termed sister chromatids, from S-phase until anaphase onset. However, the mechanism that first pairs sister chromatids together remains unknown. CTF7/ECO1 is the founding member of a cohesion establishment pathway and appears to couple sister chromatid pairing reactions to DNA replication. Sister pairing can also be induced outside of S-phase, suggesting that Ctf7p establishment activity is tightly regulated. Currently, Ctf7p remains the only essential establishment factor known. Recent findings confirm that Ctf7p functions during S-phase, is recruited to chromatin during S-phase and binds to numerous chromatin-associated replication factors. In Phase 1, we will use site-directed mutational analyses coupled to biochemical methodologies to identify which protein interactions are required for Ctf7p chromatin recruitment and how Ctf7p recruitment/activation is regulated. Genetic and molecular analyses will then be paired to high-resolution microscopy cohesion assays to test for the role of novel alleles in cell viability and in sister pairing reactions. In Phase 2, we will identify how Ctf7p pairs sister chromatids once recruited to chromatin. We will pursue results from our lab that Ctf7p associates with Pds5p, a cohesin regulator that is required to maintain sister pairing. Ctf7p-Pds5p binding is the only known link between the processes that establish and maintain cohesion. To address these issues, we will use biochemical and cell-cycle mapping strategies to test if Ctf7p-Pds5p binding is cell cycle specific, direct and essential. Both Ctf7p and Pds5p are post-translationally modified. Molecular and biochemical methodologies will be used to determine how Ctf7p-Pds5p establishment activity is regulated over the cell cycle, regulate binding and ultimately how Ctf7p- Pds5p affect cohesin dynamics on chromatin.