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：来自非传统模型的机制和进化见解细胞用于编码正常功能的遗传信息存在于称为染色体的线状分子上。这些染色体的尖端，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;当箍缺失时，鞋带（染色体）就会磨损并散开。巧合的是，端粒上的DNA在染色体复制过程中很难维持，而染色体复制是细胞每次分裂时都会发生的。需要许多蛋白质分子一起工作，以确保端粒DNA的正确维护。这项研究将利用各种技术来研究这些蛋白质分子如何一起工作，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的完整性。这项研究将使用一种名为Ustilago maydis的真菌进行，这种真菌很容易操作，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博士后学生，使他们能够获得未来科学职业所需的技术和批判性思维技能。这些本科生将从霍洛维茨社区学院招募，该学院主要为代表性不足的少数民族学生提供服务。培养这些学生将通过促进更多样化的科学劳动力而造福社会。端粒是保护和稳定染色体末端的特殊核蛋白结构。端粒DNA是通过一种称为端粒酶的特殊逆转录酶定期添加重复序列来维持的。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的染色体末端和双链断裂。然而，自相矛盾的是，修复蛋白在两种情况下有助于促进端粒的维持。首先，端粒酶阴性细胞可以利用异常重组/修复机制（称为ALT）将端粒重复序列添加到染色体末端。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促进端粒束的维持（例如，通过稳定停滞的分叉或克服复制障碍）。我们最近发现，这两个功能的修复蛋白在端粒可以忠实地复制在玉米黑粉菌，一个模型真菌系统研究基本的细胞过程。此外，我们发现，在美国。玉米端粒蛋白和修复因子之间的几种保守的和功能上重要的相互作用。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，以促进端粒的维护，同时抑制异常修复。这是一个尚未得到详细审查的监管水平。我们还发现在美国。玉米的两个双链端粒结合蛋白具有不同的功能和与修复蛋白的相互作用。因此，我们将通过对不同真菌中端粒和修复蛋白的比较分析来研究它们是如何共同进化的。目的和方法：1）使用结合和活性测定来确定端粒-修复蛋白相互作用的分子基础，并在体外检查相互作用如何影响这些蛋白的修复活性。2）使用遗传和基于细胞的测定来确定端粒-修复蛋白相互作用在促进端粒复制和ALT中的机制和功能。3）表征双链端粒的性质，结合蛋白同源物在芽殖酵母和黑粉菌，以确定双链端粒的进化路径，结合蛋白及其与真菌修复因子的相互作用。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的学术价值和更广泛的影响审查标准。

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.