Aging induced DNA double-strand break analysis in yeast

酵母中衰老诱导的 DNA 双链断裂分析

• 批准号: 10605475
• 负责人: Lindsey N. Power
• 金额: $ 3.66万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10605475
• 关键词: Affect; Age; Age of Onset; Aging; Cardiovascular Diseases; Cell Nucleus; Cells; Centromere; ChIP-seq; Chromatin; Chromosomal Instability; Chronic Disease; Collaborations; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Maintenance; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA replication fork; Diabetes Mellitus; Disease; Dose; Double Strand Break Repair; Doxycycline; Elderly; Etiology; Event; Frequencies; Gamma-H2AX; Genetic Models; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Heart Diseases; Individual; Laboratories; Lesion; Link; Location; Longevity; Magnetism; Malignant Neoplasms; Maps; Methods; Methyl Methanesulfonate; Modeling; Molecular; Nature; Nuclear; Nuclear Proteins; Phenotype; Population; Proteins; Proteomics; Protocols documentation; Publishing; Pulsed-Field Gel Electrophoresis; RNA Polymerase I; Ribosomal DNA; Risk Factors; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Stress; System; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Type I DNA Topoisomerases; Work; Yeasts; age related; aged; cell age; chromosome loss; cohesin; experimental study; gel electrophoresis; genome integrity; genome-wide; healthspan; helicase; human old age (65+); inducible gene expression; insight; model organism; novel; overexpression; prevent; repair enzyme; repaired; tool; yeast genome

PROJECT SUMMARY/ABSTRACT Aging is a primary risk factor for most chronic diseases. As the population of individuals over the age of 65 increases in the U.S., it is critical that we understand the molecular mechanisms that drive aging, with the goal of delaying the onset of these chronic diseases. One of the “hallmarks of aging”, genomic instability, can occur in the form of DNA double-stranded breaks (DSBs), which are lethal to cells unless resolved. In S. cerevisiae, the ribosomal DNA (rDNA) is especially unstable due to its repetitive nature, high levels of transcription, and a major replication fork block site. We are using the rDNA as a tool for studying the initiating genome instability events of aging in dividing cells. Instability at the rDNA significantly contributes to the replicative aging of yeast cells. Our lab previously demonstrated age-induced depletion of several factors that maintain rDNA stability, including Sir2 and cohesin. During early aging, chromatin association by these factors was reduced at the rDNA locus, followed by later reduction at centromeres, a combination that resulted in chromosome instability. To identify additional factors that contribute to aging-induced rDNA and chromosome instability, we performed a proteomics screen on nuclei isolated from replicatively young and moderately aged yeast cells. This screen revealed depletion of multiple proteins that control chromatin topology and remodeling, especially at the rDNA. These included topoisomerases I and II, and the DNA helicase Rrm3. Taken together, we predict a model where reduced capacity to resolve DNA torsional stress during early aging results in DSBs at the rDNA. We hypothesize that the repetitive rDNA array and accumulating extrachromosomal rDNA circles then act as a “sink” for diminished DNA stabilizers and repair enzymes later in aging, ultimately contributing to genome-wide instability. To test this model, I am using a genome-wide DSB mapping and sequencing protocol in young and progressively aged cells, focusing on hotspot identification across multiple time-points (Aim1). In preliminary experiments, I have optimized this method for yeast and confirmed the rDNA as a DSB hotspot, even in young cells. I will also look for changes in distribution across lifespan of the key proteins identified in our proteomics screen using ChIP-seq. Third, I will determine if aging sensitizes cells to DSB inducing agents. In Aim2 I will determine if DSB accumulation in early aging can be rescued by re-expressing key age-depleted factors using a titratable, doxycycline-inducible overexpression system. Second, I will determine if re-expression of the candidate age-depleted proteins reduces rDNA stability or extends replicative lifespan. These experiments will give insight into how genomic instability acts as a driver for the initiating events of aging.

项目总结/摘要 衰老是大多数慢性病的主要危险因素。65岁以上的人口 美国的增长，关键是我们要了解驱动衰老的分子机制， 延缓这些慢性疾病的发作。“衰老的标志”之一，基因组不稳定性， 以DNA双链断裂（DSB）的形式存在，除非得到解决，否则对细胞是致命的。In S.酿酒酵母， 核糖体DNA（rDNA）由于其重复性质、高水平转录和 主复制分叉块站点。我们正在使用rDNA作为研究起始基因组不稳定性的工具 分裂细胞中的衰老事件。rDNA的不稳定性对酵母的复制老化有重要作用 细胞我们的实验室以前证明了年龄诱导的几种维持rDNA稳定性的因子的消耗， 包括Sir 2和粘附素。在衰老早期，这些因素引起的染色质缔合在衰老早期减少， rDNA位点，随后在着丝粒处减少，这种组合导致染色体不稳定。 为了确定导致衰老诱导的rDNA和染色体不稳定性的其他因素，我们进行了 一个蛋白质组学筛选细胞核分离的复制年轻和中度老化的酵母细胞。此屏幕 揭示了控制染色质拓扑结构和重塑的多种蛋白质的消耗，特别是在rDNA处。 这些包括拓扑异构酶I和II，以及DNA解旋酶Rrm 3。综合起来，我们预测一个模型 其中在早期老化期间降低的分解DNA扭转应力的能力导致rDNA处的DSB。我们 假设重复的rDNA阵列和积累的染色体外rDNA环，然后作为一个 在衰老后期，DNA稳定剂和修复酶的减少会“下沉”，最终导致全基因组 不稳定 为了测试这个模型，我使用了一个全基因组的DSB定位和测序协议，在年轻的， 逐渐老化的细胞，重点是跨多个时间点的热点识别（Aim 1）。初步 实验中，我已经优化了这种方法的酵母，并确认rDNA作为DSB热点，即使在年轻的 细胞我还将寻找在我们的蛋白质组学中确定的关键蛋白质在整个生命周期中的分布变化 使用ChIP-seq进行筛选。第三，我将确定衰老是否使细胞对DSB诱导剂敏感。在AIM 2中，我将 确定是否可以通过重新表达关键的年龄耗竭因子来挽救早期衰老中的DSB积累， 一种可滴定的强力霉素诱导的过表达系统。第二，我将决定是否重新表达 候选的老化蛋白降低rDNA稳定性或延长复制寿命。这些实验将 让我们深入了解基因组的不稳定性是如何作为一个驱动程序的启动事件的老化。