No abstract

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is colocalized, genome-wide, with transcribing RNA polymerase (RNAP). Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, colocalization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched upstream (within up to 12-15 kb) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP:EcTopoI interaction by either overexpression of EcTopoI competitor (CTD or inactive EcTopoI Y319F mutant) or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, uncoupling of the RNAP:EcTopoI interaction leads to R-loops accumulation genome-wide, indicating that this interaction is required for prevention of R-loops formation.