The role of establishment factors in cohesion

建立因素在凝聚力中的作用

• 批准号: 7808796
• 负责人: Jerard Hurwitz
• 金额: $ 34.35万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808796
• 关键词: Acetylesterase; Anaphase; Aneuploidy; Binding; Cell Survival; Cell division; Cells; Chromatids; Chromatin; Chromosomal Loss; Chromosome abnormality; Complex; DNA; DNA biosynthesis; Defect; Deposition; Development; Event; Failure; Genes; Genetic; Genome; Human; In Vitro; Investigation; Laboratories; Lead; Malignant Neoplasms; Modeling; Nature; Peptide Hydrolases; Phase; Play; Pre-Replication Complex; Process; Property; Protein Acetylation; Proteins; Reaction; Role; Sister; Sister Chromatid; Structure; System; Time; Yeasts; cohesin; cohesion; daughter cell; helicase; origin recognition complex; prevent; protein complex; separase

DESCRIPTION (provided by applicant): The equal distribution of the replicated genome to daughter cells is essential for cell survival. Failure to accomplish this step results in aneuploidy leading to decreased viability in yeast and chromosomal loss, developmental defects and malignancies in humans. In order to prevent such chromosomal aberrations, cells have evolved a conserved mechanism to regulate this process. Chromatids are bound together from the time of their duplication in S-phase until their equal distribution between daughter cells during anaphase. Though sister chromatid pairing and partition to daughter cells are under intensive investigation in many laboratories, little is known how sister chromatid cohesion is established. Genetic studies in yeast have identified a number of gene products essential for sister chromatid cohesion. These genes are divided into three groups: structural factors, made up of the four-subunit cohesin complex (Smc1, Scc1, Smc3 and Scc3), which holds sister chromatids together after replication. The cohesin connection to chromatids is severed through the cleavage of the Scc1 subunit by the protease separase during cell division; deposition factors, which include the proteins Scc2 and Scc4 that load cohesin onto DNA and Pds5 which plays a role in maintaining the chromatin-bound cohesin and establishment factors, Ctf7, Ctf4, Ctf18, Dcc1, Ctf8 and ChlR1, which interact with many replication components and function during S-phase. We plan to: A) develop an in vitro system capable of loading cohesin onto DNA. For this purpose, we will isolate the deposition factors Scc2p and Scc4p, examine their interactions with cohesin and determine whether this results in the opening of the cohesin ring and its loading on DNA. The putative role of the pre- replication complex in this reaction will be studied; B) further characterize the in vitro properties of the establishment proteins Ctf18-Dcc1-Ctf8-RFC2-5 (a PCNA clamp loader), Ctf7p (a PCNA interacting protein and acetylase), ChlR1 p (a 5'-3' DNA helicase) and Ctf4p (interacts with Pol alpha). We plan to explore their action in supporting DNA synthesis with DNA substrates that mimic fork-like structures. These studies are directed at exploring a model in which these proteins, all of which appear to be associated with the replication fork, act jointly with other replication factors to remodel the fork and allow its passage through the large cohesin ring.

描述(由申请人提供)：复制的基因组均匀分布到子代细胞是细胞生存的关键。未能完成这一步骤会导致非整倍体，导致酵母活力下降，并导致人类染色体丢失、发育缺陷和恶性肿瘤。为了防止这种染色体异常，细胞进化出一种保守的机制来调节这一过程。染色单体从S期开始复制，直到后期均匀分布在子细胞之间，一直结合在一起。尽管许多实验室正在对姐妹染色单体配对和子代细胞的分裂进行深入的研究，但对姐妹染色单体凝聚力的建立知之甚少。酵母中的遗传学研究已经确定了一些对姐妹染色单体凝聚力至关重要的基因产物。这些基因分为三组：结构因子，由四个亚单位粘附素复合体(Smc1、Scc1、Smc3和Scc3)组成，它在复制后将姐妹染色单体结合在一起。粘附素与染色单体的连接是通过细胞分裂过程中的蛋白酶分离酶切割Scc1亚基来切断的；沉积因子，包括将粘附素加载到DNA上的蛋白质Scc2和Scc4，以及在维持染色质结合的粘附素和建立因子方面起作用的Pds5，Ctf7、Ctf4、Ctf18、Dc1、Ctf8和Chlr1，它们在S期与许多复制组件和功能相互作用。我们计划：a)开发一种能够将粘附素装载到DNA上的体外系统。为此，我们将分离沉积因子Scc2p和Scc4p，检查它们与粘附素的相互作用，并确定这是否导致粘附素环的打开及其在DNA上的负载。将研究复制前复合体在该反应中的假定作用；b)进一步表征建立蛋白Ctf18-Dcc1-Ctf8-Rfc2-5(增殖细胞核抗原夹持器)、Ctf7p(增殖细胞核抗原相互作用蛋白和乙酰基酶)、ChlR1p(5‘-3’DNA解旋酶)和Ctf4p(与Polα相互作用)的体外性质。我们计划探索它们在用模仿叉状结构的DNA底物支持DNA合成方面的作用。这些研究旨在探索一种模型，在该模型中，这些似乎都与复制叉子相关的蛋白质与其他复制因子共同作用，重塑复制叉子，并允许其通过大的粘附素环。