Single-molecule imaging of DNA gyrase activity in living Escherichia\n coli

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and\nrelaxes positive supercoils introduced by replication and transiently by\ntranscription. Removal of these positive supercoils is essential for\nreplication fork progression and for the overall unlinking of the two duplex\nDNA strands, as well as for ongoing transcription. To address how gyrase copes\nwith these topological challenges, we used high-speed single-molecule\nfluorescence imaging in live Escherichia coli cells. We demonstrate that at\nleast 300 gyrase molecules are stably bound to the chromosome at any time, with\n~12 enzymes enriched near each replication fork. Trapping of reaction\nintermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell\ntimes of ~2 s were observed for the dispersed gyrase molecules, which we\npropose maintain steady-state levels of negative supercoiling of the\nchromosome. In contrast, the dwell time of replisome-proximal molecules was ~8\ns, consistent with these catalyzing processive positive supercoil relaxation in\nfront of the progressing replisome.\n