Investigating the Impact of Telomere Specific Oxidative Base Damage in Cellular Aging

项目摘要/摘要 端粒在细胞衰老和癌变之间的界面上起着至关重要的作用，因为它们 对氧化应激高度敏感。氧化应激，或过量的自由基，是DNA的一个普遍来源 人类受到正常新陈代谢、身体压力、污染等外部来源和 太阳辐射，以及免疫细胞信号的副产品。而端粒磨损和氧化之间的联系 应激存在，目前还没有关于端粒DNA直接氧化碱基损伤如何影响正常的报道 细胞生物学。我们团队开发了第一个诱导常见氧化损伤8-氧鸟嘌呤的系统 (8oxoG)通过将荧光素激活肽(FAP)与端粒结合蛋白融合而在端粒特异性 TRF1.我们已经建立了在端粒有均一FAP-TRF1表达的克隆细胞系，并且 发现在单次诱导端粒8oxoG后，正常细胞表现出生长停滞，这是我们没有观察到的 在两种癌细胞系中。这项研究的假设是端粒8oxoG足以产生DDR 在正常细胞中，诱导生长停滞与端粒缩短无关，这依赖于p53/p21 信号轴。此外，在诱导端粒8oxoG后，我们观察到端粒8oxoG的数量增加。 微核最早在治疗后一天出现。这个项目还将解决这些微核是如何形成的， 如果它们依赖于DNA复制，如果它们被cGAS/STIN轴感知到。这是一场竞争 该项目将首次描绘端粒氧化应激对细胞结果的贡献 与衰老和癌症有关。这项工作将普遍适用于所有人类，但也将告知 与氧化应激水平升高有关的与年龄相关的疾病的生物学，并可能促进 这一奖学金还允许获得与儿童基金会目标相关的新技能 项目，并为每个项目列出了具体的导师。这包括端粒的单分子分析。 与免疫相关的信号分子的复制、活细胞成像、RNA测序和检测 系统。此外，职业发展也是该奖学金的一个重点。它包括参加研讨会的计划 与实验技术和科学写作有关，除了参加国内和国际会议 会议介绍这一研究项目的成果。

Project Summary/Abstract Telomeres play an essential role at the interface between cellular aging and carcinogenesis because they are highly sensitive to oxidative stress. Oxidative stress, or the excess of free radicals, is a ubiquitous source of DNA damage humans experience from normal metabolism, physical stress, exogenous sources such as pollution and solar radiation, and by-products of immune cell signaling. While a link between telomere attrition and oxidative stress exists, there have been no reports on how direct oxidative base damage to telomeric DNA impacts normal cell biology. Our group has developed the first system to induce the common oxidative lesion 8-oxo-guanine (8oxoG) specifically at telomeres by fusing a fluorogen activated peptide (FAP) to the telomere binding protein TRF1. We have generated clonal cell lines that have homogenous FAP-TRF1 expression at the telomere, and find that after a single induction of telomeric 8oxoG, normal cells exhibit a growth arrest, which we did not observe in two cancer cell lines. The hypothesis of this fellowship is that telomeric 8oxoG is sufficient to produce a DDR in normal cells that induces growth arrest, independent of telomere shortening, that depends on the p53/p21 signaling axis. Moreover, following induction of telomeric 8oxoG we observe an increase in the number of micronuclei as early as one day after treatment. This project will also address how these micronuclei are forming, if they are dependent on DNA replication, and if they are sensed by the cGAS/STING axis. Competition of this project will delineate for the first time the contribution of telomeric oxidative stress to cellular outcomes related to aging and cancer. This work will have general applications to all humans, but will also inform the biology of age-related diseases that are associated with increased levels of oxidative stress, and may promote health-span in either case.This fellowship also allows for the acquisition of new skills related to the aims of the project, and has outlines specific mentors for each. This includes single molecule analyses of telomere replication, live-cell imaging, RNA-sequencing, and detection of signaling molecules related to the immune system. Moreover, career development is also a focus of this fellowship. It includes plans to attend workshops related to experimental techniques and scientific writing, in addition to attending national and international conferences to present the findings of this research project.