Project Summary The helical nature of double stranded DNA (dsDNA) innately promotes the generation of torsional stress during essential processes such as replication and transcription. In particular, replication will inevitably generate DNA supercoiling which may braid or intertwine daughter DNA strands, creating precatenanes. Such intertwining, if not properly resolved, results in DNA damage, genome instability, and replication arrest. Though critical to cellular viability, these problems are highly complex, posing significant barriers to experimentation. Thus our mechanistic understanding has remained conspicuously limited. The work proposed here aims to address the role that the torsional mechanical properties of chromatin play in determining the formation and resolution of topological impasses, and in turn, the implications this has for topoisomerase function. Aim 1 will establish methods to create, benchmark, and manipulate both single and braided chromatin substrates. Since little is known about how topoisomerases interact with chromatin substrates, Aim 2 will develop a novel assay to monitor topoisomerase activity, in real time, and directly apply this approach to examine different topoisomerases to determine their effectiveness in supercoiling removal and substrate preferences. This will also allow investigation into how therapeutic agents impact topoisomerase activity. Finally, Aim 3 will examine broadly replication generated torsion and topoisomerase binding sites genome wide. To pursue these aims we will leverage state-of-the-art single molecule and genome wide techniques – including established approaches and novel assays. The proposed research will demonstrate the broad role of the intrinsic mechanical properties of chromatin in fundamental biological processes and will have far-reaching impacts into the treatment of human disease and the development of novel therapeutic agents.