Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Abstract

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome

“…Most of our knowledge on the control of topological homeostasis by topoisomerases comes from plasmid DNA topology analyses by 1D or 2D gel electrophoresis or electron or AFM microscopy ( 62 ). Recently, molecular genomics methods were implemented to map topoisomerase activities or binding on chromosome ( 63 ). Hi-C is a popular method to study chromosome conformation, and a derivative to track inter sister contacts was recently designed in eukaryotes ( 51 ).…”

Genomic contacts reveal the control of sister chromosome decatenation in E. coli

“…Most of our knowledge on the control of topological homeostasis by topoisomerases comes from plasmid DNA topology analyses by 1D or 2D gel electrophoresis or electron or AFM microscopy (Cebrián et al, 2015). Recently, molecular genomics methods were implemented to map topoisomerase activities or binding on chromosome (McKie et al, 2020). Hi-C is a popular method to study chromosome conformation, and a derivative to track intersister contacts was recently designed in eukaryotes (Oomen et al, 2020).…”

Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States

“…Due to advancement in next-generation sequencing (NGS) this has recently become possible. NGS has revolutionized the landscape of genetic research by allowing for millions of strands to be simultaneously sequenced by the means of cell-free library preparation, making the process more effective and comprehensive [80,81]. The range of applications for NGS in research and in diagnostics is boundless, beginning with the ability to recognize mutations in disease and extending toward identifying sites of DNA-protein interactions as well as DNA break spots [82].…”

“…For instance, high-throughput, genome-wide, translocation sequencing (HTGTS) can detect translocation sites [100][101][102], global run-on and sequencing [103] (GRO-seq) recognizes active transcriptional regulatory elements. In addition, Spo11-oligo-seq, CC-seq and Topo-seq all map topoisomerase cleavage sites [81].…”