The physical and functional interplay between telomere and repair proteins: mechanistic and evolutionary insights from an unconventional model

端粒和修复蛋白之间的物理和功能相互作用：来自非常规模型的机械和进化见解

• 批准号: 1817331
• 负责人: Neal Lue
• 金额: $ 79.6万
• 依托单位: Joan and Sanford I. Weill Medical College of Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817331&HistoricalAwards=false
• 关键词: physical; functional; interplay; between; telomere

The physical and functional interplay between telomere and repair proteins: mechanistic and evolutionary insights from an unconventional modelThe genetic information that the cell uses to encode normal functions resides on thread-like molecules called chromosomes. The tips of these chromosomes, named telomeres, play especially important roles in protecting the integrity of the genetic information. Telomeres can be likened to aglets, the metal or plastic caps at the tips of shoelaces; when the aglets are missing, the shoelaces (chromosomes) become frayed and fall apart. Paradoxically, the DNA at telomeres is difficult to maintain during the process of chromosome duplication, which occurs each time a cell divides. Many protein molecules, working together are required to ensure the proper maintenance of telomere DNA. This research will utilize a variety of techniques to investigate how these protein molecules work together, how they interact with one another, and how these interactions are regulated to promote telomere integrity. The study will be carried out using a fungus called Ustilago maydis, which is easy to manipulate and has telomere features that are shared by many animals. This research will also train many undergraduate students and a postdoctoral student, allowing them to acquire the technical and critical thinking skills necessary for future careers in science. The undergraduates will be recruited from Hostos Community College, an institution that serves primarily under-represented minority students. Training these students will benefit society by promoting a more diverse scientific workforce.Telomeres are specialized nucleoprotein structures that protect and stabilize the ends of chromosomes. Telomere DNA is maintained through periodic addition of a repetitive sequence by a special reverse transcriptase named telomerase. A key function of the telomere nucleoprotein complex is to allow the cells to distinguish normal chromosome ends from double strand breaks by suppressing inappropriate DNA repair. However, repair proteins paradoxically help to promote telomere maintenance in two circumstances. First, telomerase-negative cells can utilize an aberrant recombination/repair mechanism (called ALT) to add telomere repeat tracts onto chromosome ends. Second, in telomerase-positive cells, multiple recombination repair factors promote the maintenance of telomere tracts by enhancing telomere replication (e.g., by stabilizing stalled forks or surmounting replication barriers). We showed recently that both of these functions of repair proteins at telomeres can be faithfully reproduced in Ustilago maydis, a model fungal system for studying fundamental cellular processes. Moreover, we discovered in U. maydis several conserved and functionally significant interactions between telomere proteins and repair factors. Consequently, we are poised to address how the direct physical interactions between telomere and repair proteins channel the enzymatic activity of repair factors to promote telomere maintenance while suppressing aberrant repair. This is a level of regulation that has not received detailed examinations. We have also uncovered in U. maydis two duplex telomere binding proteins with distinct functions and interactions with repair proteins. We will therefore examine how the telomere and repair proteins co-evolve through comparative analysis of these factors in different fungi.Objectives and Methods:1) Use binding and activity assays to define the molecular basis of telomere-repair protein interactions and examine how the interactions affect the repair activities of these proteins in vitro.2) Use genetic and cell-based assays to determine the mechanisms and function of the telomere-repair protein interactions in promoting telomere replication and ALT in vivo.3) Characterize the properties of duplex telomere-binding protein homologs in budding yeast and Ustilago to determine the evolutionary path of duplex telomere-binding proteins and their interaction with repair factors in fungi.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

端粒和修复蛋白之间的物理和功能相互作用：来自一个非常规模型的机械和进化的见解细胞用来编码正常功能的遗传信息存在于被称为染色体的线状分子上。这些染色体的尖端，被称为端粒，在保护遗传信息的完整性方面起着特别重要的作用。端粒可以被比作鞋带末端的金属或塑料帽；当肩带丢失时，鞋带（染色体）就会磨损并脱落。矛盾的是，在每次细胞分裂时发生的染色体复制过程中，端粒上的DNA很难维持。许多蛋白质分子协同工作，以确保端粒DNA的适当维护。这项研究将利用各种技术来研究这些蛋白质分子如何协同工作，它们如何相互作用，以及如何调节这些相互作用以促进端粒完整性。这项研究将使用一种名为Ustilago maydis的真菌进行，这种真菌易于操作，并且具有许多动物共有的端粒特征。这项研究还将培养许多本科生和一名博士后，使他们获得未来科学事业所需的技术和批判性思维技能。这些本科生将从霍斯托斯社区学院（Hostos Community College）招募，该学院主要招收少数族裔学生。培训这些学生将通过促进更多样化的科学劳动力来造福社会。端粒是保护和稳定染色体末端的特殊核蛋白结构。端粒DNA是通过一种叫做端粒酶的特殊逆转录酶周期性地添加一个重复序列来维持的。端粒核蛋白复合物的一个关键功能是通过抑制不适当的DNA修复，使细胞能够区分正常染色体末端和双链断裂。然而，在两种情况下，修复蛋白矛盾地帮助促进端粒的维持。首先，端粒酶阴性细胞可以利用异常重组/修复机制（称为ALT）将端粒重复束添加到染色体末端。其次，在端粒酶阳性细胞中，多种重组修复因子通过增强端粒复制（例如，通过稳定停滞的分叉或克服复制障碍）来促进端粒束的维持。我们最近表明，端粒修复蛋白的这两种功能都可以在黑穗病菌（Ustilago maydis）中忠实地复制，黑穗病菌是研究基本细胞过程的模型真菌系统。此外，我们在美国maydis中发现了端粒蛋白和修复因子之间的一些保守的和功能显著的相互作用。因此，我们准备解决端粒和修复蛋白之间的直接物理相互作用如何引导修复因子的酶活性，以促进端粒维护，同时抑制异常修复。这是一个尚未接受详细审查的监管水平。我们还发现了两种具有不同功能和与修复蛋白相互作用的双端粒结合蛋白。因此，我们将通过对不同真菌中这些因素的比较分析来研究端粒和修复蛋白如何共同进化。目的和方法：1)利用结合和活性实验确定端粒-修复蛋白相互作用的分子基础，并研究这些相互作用如何影响这些蛋白的体外修复活性。2)利用遗传学和基于细胞的方法确定端粒-修复蛋白相互作用在体内促进端粒复制和ALT的机制和功能。3)研究出芽酵母和黑穗菌双端粒结合蛋白同源物的特性，确定双端粒结合蛋白在真菌中的进化路径及其与修复因子的相互作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。