Determining the effects of ribonucleotides on telomere integrity

项目摘要/摘要 核糖核酸(Ribos)是由dna聚合酶错误插入的，最近被认为是最多的 大多数基因组都有常见的病变。与脱氧核糖核苷酸相比，核糖核酸多了一个2‘-羟基 这使得它们更容易自发水解。此外，未修复的Ribos会提升 基因组不稳定，并与各种人类疾病有关。与基因组核糖体相比，其影响 核糖体在端粒中的分布尚不清楚。端粒是位于端粒末端的保护性非编码DNA结构 维持基因组完整性所必需的染色体，如端粒的作用 在无数的人类疾病中出现功能障碍。由于缺乏选择性地扰乱肋骨插入率的工具 对于端粒，端粒核糖体的流行和影响仍未被探索。为此，我们有 通过发现一种缺乏端粒酶的变异端粒酶，为研究端粒核糖体的作用开发了解决方案 任何核糖体识别活动，并开发一种测定端粒核糖体水平的方法。 此外，我已经确定了两个与端粒酶疾病相关的变异体(A716V和Y717N)，它们似乎 扰乱端粒酶核糖体歧视。这项建议的总体目标是确定水平和 端粒核糖核酸酶的作用，以及端粒核糖核酸酶插入增加和人类 疾病。我假设核糖核苷酸存在于端粒中，并且增加核糖核酸插入促进 端粒脱帽和基因组不稳定。为了测试这一点，我提出了以下目标：1)表征动力学 和端粒酶疾病相关变异体的结构效应以及2)决定每个变异体如何起作用 端粒完整性和细胞活力。为了实现这些目标，我将描述预稳态动力学 用端粒酶同源物赤霉菌端粒酶逆转录酶鉴定A716V和Y717N (TcTERT)(Aim 1A)，以及使用人端粒酶来确定端粒酶活性的差异(Aim 1B)。我将通过表征A716V和Y717N的结构重排来补充动力学分析 在TcTERT中使用X射线结晶学(目标1C)。在全面的生化特征之后 每个变体，我将确定表达每个端粒酶变体(AIM)的细胞系中端粒核糖体的水平 2a)。最后，将使用免疫荧光显微镜来确定增加的端粒核糖体的影响。 识别端粒功能障碍引起的病灶(Aim 2B)，这是端粒DNA的常用标记 损坏反应。这一创新项目将在堪萨斯大学医学中心完成 专家赞助团队的监督。除了技术技能，培训计划还优先考虑 科学交流和指导方面的培训。拟议的实验将为我提供 为获得成功的博士后奖学金，并最终成为富有成效的独立研究事业奠定了基础。

Project Summary/Abstract Ribonucleotides (ribos) are misinserted by DNA polymerases and have recently been recognized as the most common lesion across most of the genome. Compared to deoxyribonucleotides, ribos have an additional 2'-OH that makes them much more susceptible to spontaneous hydrolysis. Additionally, unrepaired ribos promote genomic instability and are associated with various human diseases. In comparison to genomic ribos, the impact of ribos in telomeres is unknown. Telomeres are protective noncoding DNA structures at the ends of chromosomes that are essential for maintaining genomic integrity, as highlighted by the role of telomere dysfunction in a myriad of human diseases. Due to the lack of a tool to selectively perturb ribo insertion rates into telomeres, the prevalence, and effects of telomeric ribos remains unexplored. To this end, we have developed solutions for investigating the role of telomeric ribos by discovering a variant telomerase that lacks any ribo discrimination activity and by developing an assay to determine the levels of telomeric ribos. Furthermore, I have identified two telomerase disease associated variants (A716V and Y717N) which appear to perturb telomerase ribo discrimination. The overarching goal of this proposal is to determine the levels and effects of telomeric ribos, as well as establish a causal role for increased telomeric ribo insertion and human disease. I hypothesize that ribonucleotides are present in telomeres and that increased ribo insertion promotes telomere uncapping and genomic instability. To test this, I propose the following aims: 1) Characterize the kinetic and structural effects of telomerase disease-associated variants and 2) Determine how each variant effects telomere integrity and cell viability. To accomplish these aims I will characterize the pre-steady state kinetics of A716V and Y717N using the telomerase homolog Tribolium castaneum telomerase reverse transcriptase (tcTERT) (Aim 1A), as well as use human telomerase to determine differences in telomerase processivity (Aim 1B). I will complement the kinetic analysis by characterizing the structural rearrangements of A716V and Y717N in tcTERT using X-ray crystallography (Aim 1C). Following the comprehensive biochemical characterization of each variant, I will determine the levels of telomeric ribos in cell lines expressing each telomerase variant (Aim 2A). Finally, the effects of increased telomeric ribos will be determined using immunofluorescence microscopy to identify telomere dysfunction induced foci (Aim 2B), which are a commonly used marker of a telomere DNA damage response. This innovative project will be completed at the University of Kansas Medical Center under the supervision of an expert sponsorship team. In addition to technical skills, the training plan also prioritizes training in scientific communication and mentorship. The proposed experiments will provide me with the foundation for a successful post-doctoral fellowship and, ultimately, a productive independent research career.