Mapping E. coli Topoisomerase IV binding and activity sites

Abstract

This methods article described a protocol aiming at mapping E. coli Topoisomerase IV (Topo IV) binding and cleavage activity sites on the genome. The approach is readily applicable to any Type II topoisomerase on a broad variety of gram-positive and gram-negative bacterial species. Conventional ChIP-seq of flag tagged Topo IV subunits and a novel method aimed at trapping only DNA bound to active Topo IV (called NorfliP) are described. NorfliP relies on the ability of norfloxacin, a quinolone drug, to cross-link the 5′ ends of the DNA breaks with the catalytic tyrosine of bacterial Type II topoisomerases. These methods give complementary results and their combination brought important insights on both the function and regulation of Topo IV.