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: 10352434
• 负责人: NEAL F LUE
• 金额: $ 39.05万
• 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/10352434
• 关键词: Address; Area; BRCA2 gene; Binding; Biochemical; Biochemistry; Biological Assay; Bone marrow failure; 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; 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.

项目总结/摘要 端粒是位于真核生物染色体末端的核蛋白结构， 基因组的稳定性端粒DNA是由一个短重复序列的大量拷贝组成的， 这是由于（1）末端复制问题阻止了亲本DNA的完全复制;以及（2） 端粒DNA和染色质形成复制屏障的倾向。帮助克服困难的主要参与者 这些困难包括（1）端粒酶，一种特殊的逆转录酶，它将“G-链”重复序列添加到3'端， （2）primase-Pol a（PP），在染色体的5'端添加“C-链”重复序列; 和（3）通过端粒促进半保守复制的解旋酶和修复蛋白。端粒酶 已经进行了详细的调查，对它的机制和调节了解很多。因此，我们认为， 在本申请中，我们将重点关注primase-Pol a和修复蛋白如Rad 51和Brh 2的作用 （BRCA2）。该研究将采用两种真菌模型（光滑念珠菌和玉米黑粉菌），每种模型都有自己的 独特的优势 在目的1中，我们将研究PP的机制和CST的调节，端粒结合复合物。我们 已经确定了CST和PP的Stn 1和Pol 12亚基之间的关键和保守界面， 表明这种相互作用可能触发PP中的构象转换以促进DNA合成。我们将 使用生物化学、cyroEM和smFRET的组合来解决这种新的构象转换机制。 此外，CST和PP都与端粒复制和全基因组复制应激有关 反应，虽然基本机制知之甚少。因此，我们将剖析 CST-PP相互作用在这些途径。这些研究将使用C。glabrata蛋白质，因为它们 很容易提纯，也易于生化处理在目标2 - 3中，我们将讨论两个核心修复的机制 蛋白质（Rad 51和Brh 2 [BRCA 2]）在端粒复制和端粒帽。我们开发了一个高- 端粒复制缺陷的分辨率测定，并使用该测定来证明几种端粒复制缺陷的关键功能。 修复蛋白我们还发现了一个新的和保守的相互作用Rad 51端粒蛋白 Pot 1，这表明新的端粒特异性调节机制。因此，在这两个目标，我们将剖析 Rad 51在端粒中的作用机制，并确定其功能如何通过Pot 1和Brh 2调节， 遗传学和生物化学的结合因为RAD 51和BRCA 2因子也与 促进整个基因组的复制和稳定停滞的叉，我们的工作可能会导致一个更完整的 从他们的机制来看。这项调查将使用玉米黑粉菌进行，因为与标准 真菌、U.玉米在重组和端粒方面与哺乳动物具有高度的相似性， 机械.