Determining the effects of ribonucleotides on telomere integrity

项目总结/摘要 核糖核苷酸（ribos）被DNA聚合酶错误插入，并且最近被认为是最重要的DNA聚合酶。 在大多数基因组中常见的病变。与脱氧核糖核苷酸相比，核糖具有额外的2 '-OH 使它们更容易自发水解。此外，未修复的核糖促进 基因组不稳定性，并与各种人类疾病有关。与基因组核糖相比， 端粒中的核糖是未知的。端粒是位于染色体末端的保护性非编码DNA结构， 染色体是维持基因组完整性所必需的，端粒的作用突出了这一点 在无数人类疾病中的功能障碍。由于缺乏选择性干扰ribo插入率的工具， 然而，端粒核糖的普遍性和作用仍然没有被探索。为此我们 通过发现一种缺少端粒的变异端粒酶， 任何核糖识别活性，并通过开发测定端粒核糖水平的测定。 此外，我已经鉴定了两种端粒酶疾病相关的变体（A716 V和Y 717 N），它们似乎 干扰端粒酶核糖体识别。本提案的总体目标是确定 端粒核糖的影响，以及建立一个因果关系的作用，增加端粒核糖插入和人类 疾病我假设核糖核苷酸存在于端粒中，核糖插入的增加促进了端粒的形成。 端粒脱帽和基因组不稳定性。为了验证这一点，我提出了以下目标：1）表征动力学 和端粒酶疾病相关变体的结构效应，以及2）确定每种变体如何影响 端粒完整性和细胞活力。为了实现这些目标，我将表征的前稳态动力学的 A716 V和Y 717 N使用端粒酶同源物赤拟谷盗端粒酶逆转录酶 （tcTERT）（Aim 1A），以及使用人端粒酶来确定端粒酶持续合成能力的差异（Aim 1B）。我将通过表征A716 V和Y 717 N的结构重排来补充动力学分析 在tcTERT中使用X射线晶体学（Aim 1C）。根据全面的生化表征， 每种变体，我将确定表达每种端粒酶变体的细胞系中端粒核糖的水平（目的 2A）。最后，端粒核糖增加的影响将使用免疫荧光显微镜来确定 鉴定端粒功能障碍诱导的病灶（Aim 2B），其是端粒DNA的常用标记物， 损伤响应这个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。拟议中的实验将为我提供 成功的博士后奖学金的基础，并最终，一个富有成效的独立研究生涯。

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.