BioProject

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. More...

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seq’s exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome. Overall design: Supercoiled pBR322 DNA was cleaved with E. coli gyrase or topoisomerase IV and spiked with three DNA oligonucleotides. The DNA was subject to standard DNA-seq library preparation to label the enzyme-cleaved ends, fragmented, and subject to a second round of standard DNA-seq library preparation to label the fragmented ends. The labelled DNA was pair-end sequenced and mapped to the plasmid and spike-in oligonucleotide reference sequences. The reads from the topo-cleaved plasmid DNA ends were normalized by the reads from the spike-in ends and were used to determine the gyrase or topo IV cleavage levels at each position on the plasmid. Less...