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.

项目概要/摘要 衰老是大多数慢性病的主要危险因素。随着 65 岁以上人口的 在美国，了解导致衰老的分子机制至关重要，目标是 延缓这些慢性疾病的发生。 “衰老的标志”之一，基因组不稳定，可能会发生 以 DNA 双链断裂 (DSB) 的形式出现，除非解决，否则对细胞是致命的。在酿酒酵母中， 核糖体 DNA (rDNA) 由于其重复性、高水平转录和 主要复制叉块位点。我们使用 rDNA 作为研究起始基因组不稳定性的工具 分裂细胞的衰老事件。 rDNA 的不稳定性显着促进酵母的复制老化 细胞。我们的实验室之前证明了年龄引起的几个维持 rDNA 稳定性的因素的损耗， 包括 Sir2 和 cohesin。在衰老早期，这些因素导致的染色质关联在 rDNA 位点，随后着丝粒减少，这一组合导致染色体不稳定。 为了确定导致衰老引起的 rDNA 和染色体不稳定的其他因素，我们进行了 对从具有复制能力的年轻和中等老化酵母细胞中分离出的细胞核进行蛋白质组学筛选。这个画面 揭示了控制染色质拓扑和重塑的多种蛋白质的耗尽，尤其是在 rDNA 处。 其中包括拓扑异构酶 I 和 II，以及 DNA 解旋酶 Rrm3。综合起来，我们预测一个模型 早期老化过程中解决 DNA 扭转应力的能力降低，导致 rDNA 处出现 DSB。我们 假设重复的 rDNA 阵列和积累的染色体外 rDNA 环然后充当 衰老后期 DNA 稳定剂和修复酶减少的“下沉”，最终促成全基因组 不稳定。 为了测试这个模型，我在年轻人和老年人中使用了全基因组 DSB 作图和测序方案。 逐渐老化的细胞，重点关注跨多个时间点的热点识别（目标1）。在初步 实验中，我针对酵母优化了这种方法，并确认 rDNA 是 DSB 热点，即使在年轻的酵母中也是如此。 细胞。我还将寻找蛋白质组学中确定的关键蛋白质在整个生命周期中分布的变化 使用 ChIP-seq 进行筛选。第三，我将确定衰老是否会使细胞对 DSB 诱导剂敏感。在《Aim2》中我会 确定是否可以通过重新表达关键的年龄消耗因子来挽救早期衰老中的 DSB 积累 可滴定的强力霉素诱导的过表达系统。其次，我将确定是否重新表达 候选年龄耗尽蛋白会降低 rDNA 稳定性或延长复制寿命。这些实验将 深入了解基因组不稳定性如何作为衰老起始事件的驱动因素。