Telomere terminal extension and replication: mechanisms and links to DNA repair

端粒末端延伸和复制：DNA 修复的机制和联系

• 批准号: 9973815
• 负责人: NEAL F LUE
• 金额: $ 40.68万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-11 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973815
• 关键词: Address; Area; BRCA2 gene; Binding; Biochemical; Biochemistry; Biological Assay; Candida glabrata; Cell division; Chromatin; Chromosomes; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Damage; DNA Primase; DNA Repair; DNA biosynthesis; Defect; Development; Disease; Enzymes; Exhibits; Genetic; Genetic Recombination; Genome; Genome Stability; Investigation; Lead; Link; Liver Fibrosis; Lung; Malignant Neoplasms; Mammals; Mediating; Modeling; Molecular; Molecular Conformation; Nucleoproteins; Pancytopenia; Pathway interactions; Play; Polymerase; Proteins; RNA-Directed DNA Polymerase; Regulation; Research; Resolution; Role; Series; Structural Models; Structure; Telomerase; Telomere Capping; Telomere Maintenance; Ustilago; Work; biological adaptation to stress; fungus; genome-wide; helicase; human disease; insight; mutant; novel; novel diagnostics; novel therapeutics; prevent; recruit; repaired; replication stress; response; single-molecule FRET; telomere

Project Summary/Abstract Telomeres, the specialized nucleoprotein structures located at the ends of eukaryotic chromosomes, are critical for genome stability. Telomere DNA, which consists of numerous copies of a short repeat, is difficult to maintain owing to (1) the end replication problem that prevents the complete duplication of parental DNA; and (2) the propensity of telomere DNA and chromatin to form replication barriers. The main players that help to overcome these difficulties include (1) telomerase, a special reverse transcriptase that adds “G-strand” repeats onto the 3’ ends of chromosomes; (2) primase-Pol a (PP), which adds “C-strand” repeats onto the 5’ ends of chromosomes; and (3) helicases and repair proteins that facilitate semi-conservative replication through telomeres. Telomerase has been subjected to detailed investigation and much is known about its mechanisms and regulation. Hence, in this application, we will focus on the roles of primase-Pol a and repair proteins such as Rad51 and Brh2 (BRCA2). The study will employ two fungal models (Candida glabrata and Ustilago maydis), each with its own unique advantages. In Aim 1, we will examine the mechanisms of PP and its regulation by CST, a telomere binding complex. We have identified a critical and conserved interface between the Stn1 and Pol12 subunits of CST and PP, and shown that this interaction likely triggers a conformational switch in PP to facilitate DNA synthesis. We will address this novel conformational switch mechanism using a combination of biochemistry, cyroEM and smFRET. In addition, both CST and PP have been linked to telomere replication and genome-wide replication stress response, though the underlying mechanisms are poorly understood. Accordingly, we will dissect the role of the CST-PP interaction in these pathways. These studies will be conducted using C. glabrata proteins because they are easily purified and biochemically tractable. In Aim 2 – 3, we will address the mechanisms of two core repair proteins (Rad51 and Brh2[BRCA2]) in telomere replication and telomere capping. we have developed a high- resolution assay for telomere replication defects and used the assay to demonstrate critical functions for several repair proteins. We have also uncovered a novel and conserved interaction between Rad51 the telomere protein Pot1, which suggests novel, telomere-specific regulatory mechanisms. Hence in these two aims, we will dissect the mechanisms of Rad51 at telomeres and determine how its functions are regulated by Pot1 and Brh2 using a combination of genetics and biochemistry. Because RAD51 and BRCA2 factors have also been implicated in promoting replication and stabilizing stalled forks throughout the genome, our work may lead to a more integrated view of their mechanisms. This investigation will be carried out using Ustilago maydis because unlike standard fungi, U. maydis exhibits a high degree of similarity to mammals with respect to the recombination and telomere machinery.

项目摘要/摘要 端粒是位于真核染色体末端的专门核蛋白结构，是关键的 用于基因组稳定性。端粒DNA由许多简短重复的副本组成，很难维护 由于（1）最终复制问题，从而阻止了父母DNA的完全重复； （2） 端粒DNA和染色质的倾向形成复制屏障。有助于克服的主要球员 这些困难包括（1）端粒酶，端粒酶，一种特殊的逆转录酶，在3'中添加“ G链”重复 染色体的末端； （2）Primase-Pol A（PP），在染色体的5'末端添加“ C链”重复； （3）促进通过端粒促进半保守复制的解旋酶和修复蛋白质。端粒酶 已经接受了详细的研究，并且对其机制和调节知之甚少。因此， 在此应用中，我们将重点介绍Primase-Pol A的作用和修复蛋白，例如RAD51和BRH2 （BRCA2）。该研究将采用两种真菌型号（Candida Glabrata和Ustilago Maydis），每种都有自己的 独特的优势。 在AIM 1中，我们将检查PP的机制及其调节，CST是一种端粒结合复合物。我们 已经确定了CST和PP的STN1和POL12亚基之间的关键和构成的接口，以及 表明这种相互作用可能触发PP中的构象开关以促进DNA合成。我们将 使用生物化学，Cyroem和Smfret的组合来解决这种新颖的会议开关机制。 此外，CST和PP都与端粒复制和全基因组复制应力有关 反应，尽管基本机制知之甚少。彼此之间，我们将剖析 这些途径中的CST-PP相互作用。这些研究将使用C. glabrata蛋白进行，因为它们 易于纯化并在生化上可进行。在AIM 2 - 3中，我们将解决两个核心维修的机制 蛋白质（RAD51和BRH2 [BRCA2]）在端粒复制和端粒上限中。我们已经开发了一个高 解决端粒复制缺陷的分辨率测定法，并使用该测定法证明了几种关键功能 修复蛋白质。我们还发现了Rad51端粒蛋白之间的小说和组成的相互作用 POT1，提出了新颖的端粒特异性调节机制。因此，在这两个目标中，我们将剖析 端粒处的RAD51的机制，并确定其功能如何通过POT1和BRH2调节 遗传学和生物化学的结合。因为Rad51和BRCA2因子也与 在整个基因组中促进复制和稳定失速的叉子，我们的工作可能会导致更加整合 他们的机制的视图。这项投资将使用Ustilago Maydis进行，因为与标准不同 U. Maydis真菌与哺乳动物相对于重组和端粒表现出很高的相似性 机械。