Biophysical, Topological, and Functional Studies of Endogenous Circular DNAs

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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.