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

项目概要/摘要 端粒是位于真核染色体末端的特殊核蛋白结构，至关重要 为了基因组的稳定性。端粒 DNA 由大量短重复序列组成，很难维持 由于（1）末端复制问题阻碍了亲代DNA的完全复制；和（2） 端粒 DNA 和染色质形成复制障碍的倾向。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，可将“G 链”重复添加到 3' 端 染色体末端； (2) primase-Pol a (PP)，它将“C链”重复添加到染色体的5’端； (3)解旋酶和修复蛋白，通过端粒促进半保守复制。端粒酶 已经进行了详细的调查，并且对其机制和监管有很多了解。因此， 在此应用中，我们将重点关注引物酶-Pol a 和修复蛋白（如 Rad51 和 Brh2）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特点 独特的优势。 在目标 1 中，我们将研究 PP 的机制及其受 CST（一种端粒结合复合物）的调节。我们 已经确定了 CST 和 PP 的 Stn1 和 Pol12 亚基之间的关键且保守的界面，并且 结果表明，这种相互作用可能会触发 PP 的构象转换，以促进 DNA 合成。我们会 使用生物化学、cyroEM 和 smFRET 的组合来解决这种新颖的构象转换机制。 此外，CST 和 PP 都与端粒复制和全基因组复制应激有关 反应，尽管其基本机制尚不清楚。因此，我们将剖析 CST-PP 在这些途径中相互作用。这些研究将使用光滑 C. glabrata 蛋白进行，因为它们 易于纯化且易于生化处理。在目标 2-3 中，我们将解决两个核心修复的机制 端粒复制和端粒加帽中的蛋白质（Rad51 和 Brh2[BRCA2]）。我们开发了一种高 端粒复制缺陷的分辨率测定，并使用该测定来证明几个关键功能 修复蛋白质。我们还发现了端粒蛋白 Rad51 之间新颖且保守的相互作用 Pot1，它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 使用 Pot1 和 Brh2 来了解 Rad51 在端粒上的机制并确定其功能如何受到 Pot1 和 Brh2 的调节 遗传学和生物化学的结合。因为 RAD51 和 BRCA2 因素也与 促进复制并稳定整个基因组中停滞的分叉，我们的工作可能会带来更加整合的结果 对其机制的看法。本次调查将使用玉米黑粉菌进行，因为与标准玉米黑粉菌不同 真菌中，玉米 U. maydis 在重组和端粒方面与哺乳动物表现出高度相似性 机械。

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.