BPS2026 – Highly sensitive mapping of E. coli gyrase and topoisomerase IV cleavage of genomic DNA in vitro

Type II topoisomerases are essential enzymes that transiently cleave double-stranded DNA. The location and frequency of this cleavage is important for topoisomerase function, genomic stability, and the action of clinically important anticancer and antibacterial drugs, e.g., quinolones. Recently, we developed a simplified high-accuracy end-sequencing (SHAN-seq) method to sensitively map cleavage complexes on plasmid DNA in vitro. Using our SHAN-seq method, we showed that E. coli gyrase and topoisomerase IV cleave at least one site every 10 bp, albeit with frequencies that vary by two-to-three orders of magnitude. Here, we have adapted our method to sensitively map topoisomerase cleavage on genomic DNA in vitro. With this updated method, we mapped E. coli gyrase and topoisomerase IV cleavage of E. coli M1655 genomic DNA both in the presence and absence of the quinolone, ciprofloxacin. We identified tens of thousands of cleavage sites across the E. coli genome, more than an order of a magnitude and more sites than previous in vivo measurements in the presence of ciprofloxacin. Based on these sites, we trained a machine learning model that accurately predicts E. coli gyrase and topoisomerase IV cleavage based on DNA sequence. Our results provide an unprecedented high-resolution and quantitative view into the intrinsic and ciprofloxacin-altered E. coli gyrase and topoisomerase IV cleavage of genomic DNA. Moreover, our results suggest that DNA sequence may play an important physiological role in localizing topoisomerases to specific genomic regions, such as transcription start and end sites.