DNA replication is stressful because unwinding of the DNA strands by the replication fork generates highly structured, supercoiled DNA that must be removed or else the replication fork will stall. DNA topoisomerases are ubiquitous enzymes that are essential for relaxing the supercoiling barriers formed during DNA replication. Although the mechanisms of how topoisomerases relieve DNA strain have been well-described, how these enzymes are regulated in vivo and how they are localized to specific regions of the genome, e.g., ahead of replication forks, remains poorly understood. As therapeutics that target topoisomerases are used to treat disorders such as cancer and bacterial infections, a better understanding of these enzymes will be key for future drug development. My postdoctoral studies identified an essential chromosome structuring protein called GapR in the bacterium Caulobacter crescentus that is required for replication and specifically recognizes overtwisted DNA, such as the DNA ahead of replication forks. Critically, GapR also stimulates the activity of bacterial topoisomerases, gyrase and topo IV, to relax highly supercoiled DNA by an unknown mechanism. This proposal will examine new regulatory paradigms by investigating how chromosome structuring proteins such as GapR control topoisomerase activity during replication. Aim 1 will elucidate the mechanisms by which GapR stimulates topoisomerase activity. I will perform co-immunoprecipitation assays to determine if GapR interacts with topoisomerases to stimulate their activities. Alternatively, or in addition, GapR could trap supercoiling by binding overtwisted DNA and consequently allow these trapped supercoils to be more efficiently relaxed by gyrase and topo IV. I will test this idea by examining how GapR interacts with DNA using magnetic tweezers. Lastly, I will use magnetic tweezers to directly assess how GapR stimulates the topoisomerase catalytic cycle. Aim 2 will examine how GapR regulates topoisomerase localization and activity to promote replication. I will determine how loss of GapR affects topoisomerase binding with ChIP-seq and develop assays to examine topoisomerase activity genome-wide by sequencing the DNA trapped in catalytically active topoisomerases. I will also assess how loss of GapR affects replication fork progression, particularly in regions that have hyper supercoiling, by examining binding of replicative helicase with ChIP-seq. In Aim 3, I will search for additional, GapR-like topoisomerase co-factors using mass spectrometry and transposon-based screens. I will subsequently characterize any identified co-factor proteins with biochemical, biophysical, and genetic approaches. The experiments within these aims will be initiated during the K99 phase of the award and will include training with magnetic tweezers to study topoisomerase mechanism and high- throughput approaches to study topoisomerase activity in vivo. Together, the experiments in this proposal will describe...