Biophysical, Topological, and Functional Studies of Endogenous Circular DNAs

内源性环状DNA的生物物理、拓扑和功能研究 (PI：S.D.Levene) 项目摘要/摘要 内源性环状DNA元件的存在，被称为染色体外环状DNA(EccDNA)，具有 在从植物到人类的各种生物中已经存在了近50年。直到最近， 人们对这些分子的功能、生物发生和序列性质知之甚少。然而，a 最近这一领域的爆炸已经开始积累证据，表明产生这些物种的机制在 在基因组动力学中的重要作用。外源基因的生物发生与DNA变异之间的关系 基因座拷贝数产生细胞异质性，这对健康和 细胞功能失调，对癌症进展和治疗有重大影响。此应用程序提供了一个 融合生物物理和基因组方法的前所未有的机会，这些方法对推进eccDNA至关重要 在近三十年的DNA拓扑和核酸物理经验的基础上研究和建立PI 化学反应。我们将使用一种创新的、多方面的方法，将生物物理和DNA拓扑学相结合- 基于DNA/染色质分离和鉴定的技术 分析。使用标准和工程化的人类细胞系，我们将定义eccDNA拓扑结构的作用(例如， 超螺旋)、序列、大小和核小体组织在内源性环状病毒的生物活性中的作用 DNA。这项工作的一个主要成果将是对eccDNA生物发生的高级量化理解， 维持、组织和生物功能。此外，我们还将确定 EccDNA元件，可用于分子治疗和其他应用，包括 诊断。

Biophysical, Topological, and Functional Studies of Endogenous Circular DNAs (PI: S.D. Levene) Project Summary/Abstract The existence of endogenous circular DNA elements, termed extrachromosomal circular DNA (eccDNA), has been established in a wide range of organisms from plants to humans for close to fifty years. Until recently, little was known about the functions, biogenesis, and sequence properties of these molecules. However, a recent explosion in the field has begun to amass evidence that the mechanisms producing these species play important roles in genome dynamics. The connection between eccDNA biogenesis and variations in DNA- locus copy number generate cellular heterogeneity, which has important implications for healthy and dysfunctional cells and major impact on cancer progression and treatment. This application offers an unprecedented opportunity to fuse biophysical and genomic approaches that are crucial to advance eccDNA research and builds on the PIs nearly thirty years of experience in DNA topology and nucleic-acid physical chemistry. We will use an innovative, multi-faceted approach that combines biophysical and DNA-topology- based techniques for DNA/chromatin isolation and characterization with leading-edge tools for genetic analysis. Using standard and engineered human cell lines, we will define the roles of eccDNA topology (e.g., supercoiling), sequence, size, and nucleosomal organization in the biological activities of endogenous circular DNAs. A major outcome of this work will be an advanced quantitative understanding of eccDNA biogenesis, maintenance, organization, and biological function. In addition, we will identify structural properties unique to eccDNA elements, which can be exploited for molecular therapeutics and other applications including diagnostics.