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: 10155499
• 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/10155499
• 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 端粒是一种特殊的核蛋白结构，位于真核细胞染色体的末端，是至关重要的。 为了基因组的稳定性。端粒DNA由许多拷贝的短重复序列组成，很难维持 由于(1)阻止亲本DNA完全复制的末端复制问题；以及(2) 端粒DNA和染色质形成复制障碍的倾向。帮助克服这些困难的主要参与者 这些困难包括：(1)端粒酶，一种特殊的逆转录酶，它在3‘端添加“G链”重复序列 (2)Primase-Pol a(PP)，它在染色体的5‘端增加“C-链”重复； 以及(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，对其机制和监管知道很多。因此， 在这个应用中，我们将重点研究Primase-Pola和修复蛋白如RAD51和Brh2的作用 (BRCA2)。这项研究将采用两种真菌模型(光滑假丝酵母和玉米黑粉菌)，每个模型都有自己的模型 优势得天独厚。 在目标1中，我们将研究PP的机制以及端粒结合复合体CST对PP的调节。我们 已经确定了CST和PP的Stn1和Pol12亚基之间的关键和保守的界面，以及 结果表明，这种相互作用可能触发PP的构象转换，以促进DNA合成。我们会 使用生物化学、CyroEM和smFRET的组合来解决这一新颖的构象开关机制。 此外，CST和PP都与端粒复制和全基因组复制压力有关 反应，尽管基本的机制还不是很清楚。因此，我们将剖析 CST-PP在这些途径中的相互作用。这些研究将使用光肩星天牛蛋白进行，因为它们 很容易提纯，而且易于生化处理。在目标2-3中，我们将讨论两个核心修复的机制 端粒复制和端粒封端中的蛋白质(RAD51和Brh2[BRCA2])。我们已经开发出了一种高度- 对端粒复制缺陷的分辨试验，并用该试验展示了几个 修复蛋白。我们还发现了端粒蛋白RAD51之间一种新颖而保守的相互作用 POT1，它提出了新的、端粒特异的调控机制。因此，在这两个目标中，我们将剖析 RAD51在端粒的作用机制以及POT1和Brh2是如何调节其功能的。 遗传学和生物化学的结合。因为RAD51和BRCA2因子也与 促进复制和稳定整个基因组中停滞不前的分叉，我们的工作可能会导致更完整的 了解它们的机制。这项调查将使用梅迪斯黑粉菌进行，因为与标准 霉菌在重组和端粒方面与哺乳动物高度相似。 机械设备。