Mechanisms of sister chromatid pairing

描述(申请人提供)：细胞必须鉴定从S期到后期的染色体复制产物，称为姐妹染色单体。然而，最初将姐妹染色单体配对在一起的机制仍然未知。CTF7/ECO1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复制结合在一起。姐妹配对也可以在S期之外被诱导，这表明Ctf7p的建立活动受到严格调控。目前，Ctf7p仍然是已知的唯一重要的建立因子。最近的研究证实，Ctf7p在S期起作用，在S期被募集到染色质，并与许多染色质相关的复制因子结合。在第一阶段，我们将使用定点突变分析结合生化方法来确定哪些蛋白质相互作用是Ctf7p染色质招募所必需的，以及Ctf7p招募/激活是如何调节的。然后，遗传和分子分析将与高分辨率显微镜凝聚力分析配对，以测试新的等位基因在细胞活力和姐妹配对反应中的作用。在第二阶段，我们将确定一旦招募到染色质，Ctf7p如何配对姐妹染色单体。我们将从我们的实验室获得Ctf7p与Pds5p相关的结果，Pds5p是维持姐妹配对所需的粘附素调节因子。Ctf7p-Pds5p结合是建立和维持凝聚力的过程之间唯一已知的联系。为了解决这些问题，我们将使用生化和细胞周期映射策略来测试Ctf7p-Pds5p结合是否是细胞周期特异的、直接的和必要的。Ctf7p和Pds5p都经过翻译后修改。分子和生化方法将被用来确定Ctf7p-Pds5p的建立活性如何在细胞周期中被调节，调节结合，并最终如何影响染色质上的粘附素动力学。

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.