Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation

Abstract

DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.

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

“…Encouraging evidence in support of the widely accepted twin-domain model, has established a greater understanding of topoisomerase activity during transcription and to a lesser extent replication. Topo II and topo I in eukaryotes exhibit higher levels of activity in the vicinity of highly transcribed genes [14,19], as has also been shown for mycobacterial topo I and gyrase [48]. Topo IV, on the other hand, demonstrates a clear preference for the dif site in E. coli [16,17], supporting the idea that decatenation is its primary cellular function [55].…”

“…This raises questions as to the determinants of topo binding and cleavage sites; how do the factors known to influence binding and activity including DNA topology and sequence play off against protein-mediated recruitment or inhibition in trans, and what other factors may influence topo binding and cleavage in vivo. It is clear the answer is not straightforward, with topo IB being directly recruited as part of the transcriptional machinery [19] and mycobacterial topo IA [48] and prokaryotic DNA gyrase [18] exhibiting more substantial DNA sequence preferences. As with all cellular processes, the mechanisms governing DNA topology maintenance are often not only protein specific but also species specific.…”

“…In 2019, another study from the same group, explored topo IA activity in more depth, contrasting binding and cleavage in Mycobacterium smegmatis, a close relative of M. tuberculosis [48]. In this study, four libraries were produced, three to probe topo IA cleavage, and another for topo IA binding.…”

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

“…[ 28–32 ] Another set of newly developed methods, generally known as “genome wide” studies, allow identification of small variations in the genome itself or the association of specific proteins, such as polymerases and topoisomerases, to specific sites along the genome. [ 33–35 ] Although we recognize the value of the data obtained using all these different methods, here we restrict ourselves to discuss data obtained using 2D gels. For a detailed and excellent review of replication fork stalling at natural impediments see ref.…”

Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA

“…1B). Mutational analysis has shown that modification of the N- or C-terminal residues of this domain in Top1 from bacterial species, including Escherichia coli, Yersinia pestis, Mycobacterium tuberculosis and Mycobacterium smegmatis , will impair DNA religation, induce an SOS response and cause cell killing (10-14).…”