Aging induced DNA double-strand break analysis in yeast

项目摘要/摘要 老龄化是大多数慢性病的主要风险因素。作为65岁以上的个人人口 在美国，随着年龄的增加，我们必须了解导致衰老的分子机制，目标是 延缓这些慢性病的发病。“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)特别不稳定，因为它具有重复性，高水平的转录，以及 主要复制分叉数据块站点。我们正在使用rdna作为研究启动基因组不稳定性的工具。 细胞分裂中的衰老事件。RDNA的不稳定性是酵母复制性老化的重要原因 细胞。我们的实验室之前证明了几种维持rDNA稳定性的因子因年龄增长而耗尽， 包括Sir2和粘附素。在衰老的早期，这些因素导致的染色质关联性在 RDNA基因座，随后是着丝粒的减少，这种组合导致了染色体的不稳定。 为了确定导致衰老诱导的rdna和染色体不稳定的其他因素，我们进行了 蛋白质组学筛选从复制年轻和中等年龄的酵母细胞中分离出的细胞核。此屏幕 揭示了控制染色质拓扑和重塑的多种蛋白质的耗尽，特别是在rDNA上。 其中包括拓扑异构酶I和II，以及DNA解旋酶Rrm3。综上所述，我们预测一个模型 其中，在早期老化过程中解决DNA扭转应力的能力降低会导致rDNA上的双链断裂。我们 假设重复的rDNA阵列和积累的染色体外rDNA环随后起到 在衰老过程中，DNA稳定剂和修复酶的数量减少，最终导致全基因组 不稳定。 为了测试这个模型，我使用了全基因组的DSB作图和测序协议 逐渐老化的细胞，专注于跨多个时间点的热点识别(Aim1)。在预赛中 实验中，我对酵母的这种方法进行了优化，并确认rdna是一个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内分布的变化 使用芯片序列的屏幕第三，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、多西环素诱导的过表达系统。其次，我将确定是否重新表达 候选的年龄耗尽的蛋白质会降低rDNA的稳定性或延长复制寿命。这些实验将 洞察基因组不稳定性如何作为衰老启动事件的驱动因素。

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.