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/文摘