The role of establishment factors in cohesion

建立因素在凝聚力中的作用

• 批准号: 7210046
• 负责人: Jerard Hurwitz
• 金额: $ 34.69万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7210046
• 关键词: Acetylesterase; Anaphase; Aneuploidy; Binding; Cell Survival; Cell division; Cells; Chromatids; Chromatin; Chromosomal Loss; Chromosome abnormality; Complex; DNA; DNA biosynthesis; DNA chemical synthesis; Defect; Deposition; Development; Endopeptidases; Event; Failure; Genes; Genetic; Genome; Human; In Vitro; Investigation; Laboratories; Lead; Malignant Neoplasms; Modeling; Nature; Numbers; PCNA gene; Peptide Hydrolases; Phase; Play; Pre-Replication Complex; Process; Property; Protein Acetylation; Proteins; Purpose; Reaction; Role; Sister; Sister Chromatid; Structure; System; Time; Yeasts; cohesin; cohesion; daughter cell; helicase; origin recognition complex; pol genes; prevent; 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细胞对于细胞存活至关重要。如果未能完成这一步骤，就会导致非整倍性，从而导致酵母活力下降、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畸变，细胞进化出了一种保守的机制来调节这一过程。染色单体从 S 期复制开始一直结合在一起，直到后期在子细胞之间均匀分布。尽管许多实验室正在深入研究姐妹染色单体配对和子细胞分配，但人们对姐妹染色单体凝聚力是如何建立的知之甚少。酵母的遗传学研究已经确定了许多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，由四个亚基粘连蛋白复合物（Smc1、Scc1、Smc3 和 Scc3）组成，在复制后将姐妹染色单体结合在一起。在细胞分裂过程中，蛋白酶分离酶会通过切割 Scc1 亚基来切断粘连蛋白与染色单体的连接；沉积因子，其中包括将粘连蛋白加载到 DNA 上的蛋白 Scc2 和 Scc4，以及在维持染色质结合粘连蛋白中发挥作用的 Pds5 和建立因子 Ctf7、Ctf4、Ctf18、Dcc1、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) 开发一种能够将粘连蛋白加载到 DNA 上的体外系统。为此，我们将分离沉积因子 Scc2p 和 Scc4p，检查它们与粘连蛋白的相互作用，并确定这是否会导致粘连蛋白环打开及其在 DNA 上的负载。将研究预复制复合物在此反应中的假定作用； B) 进一步表征建立蛋白 Ctf18-Dcc1-Ctf8-RFC2-5（PCNA 钳加载器）、Ctf7p（PCNA 相互作用蛋白和乙酰化酶）、ChlR1 p（5'-3' DNA 解旋酶）和 Ctf4p（与 Pol α 相互作用）的体外特性。我们计划探索它们在使用模仿叉状结构的 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，其中这些蛋白质（所有这些蛋白质似乎都与复制叉相关）与其他复制因子共同作用以重塑复制叉并允许其通过大粘连蛋白环。