Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is colocalized, genome-wide, with transcribing RNA polymerase (RNAP). Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, colocalization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched upstream (within up to 12-15 kb) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP:EcTopoI interaction by either overexpression of EcTopoI competitor (CTD or inactive EcTopoI Y319F mutant) or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, uncoupling of the RNAP:EcTopoI interaction leads to R-loops accumulation genome-wide, indicating that this interaction is required for prevention of R-loops formation. 

/pdf/interaction-between-transcribing-rna-polymerase-and-1jeek8ie.pdf

Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is co-localized, genome-wide, with RNA polymerase (RNAP) in transcription units. Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, co-localization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched and in and upstream (within up to 12-15 Kbs) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP-EcTopoI interaction by either overexpression of EcTopoI CTD or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, the CTD overexpression leads to R-loops accumulation genome-wide, indicating that the RNAP-EcTopoI interaction is required for prevention of R-loops formation. Article Highlights TopoI colocalizes genome-wide and interacts with RNAP in E. coli Disruption of the interaction between TopoI and RNAP decreases cells viability, leads to hypernegative DNA supercoiling, and R-loops accumulation TopoI and DNA gyrase are enriched, respectively, upstream and downstream of transcription units in accordance with twin-domain model of Liu and Wang TopoI recognizes its cleavage sites through a specific motif and by sensing negative supercoiling

https://www.biorxiv.org/content/biorxiv/early/2021/10/26/2021.10.26.465782.full.pdf

Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

E. coli type 1A topoisomerases (topos), topo I (topA) and topo III (topB) have both relaxation and decatenation activities. B. subtilis and E. coli topA topB null cells can survive owing to DNA amplifications allowing overproduction of topo IV, the main cellular decatenase that can also relax supercoiling. We show that overproducing human topo IB, a relaxase but not a decatenase, can substitute for topo IV in allowing E. coli topA null but not topA topB null cells to survive. Deleting topB exacerbates phenotypes of topA null mutants including hypernegative supercoiling, R-loop formation, and RNase HI-sensitive replication, phenotypes that are not corrected by topo IV overproduction. These phenotypes lead to Ter DNA amplification causing a chromosome segregation defect that is corrected by topo IV overproduction. Furthermore, topA topB null mutants not overproducing topo IV acquire uvrB or uvrC mutations, revealing a nucleotide excision repair (NER)-dependent problem with replication fork progression. Thus, type IA topos maintain the stability of the genome by providing essential relaxase and decatenase activities to prevent and solve topological stress related to R-loops and NER. Moreover, excess R-loop formation is well tolerated in cells that have enough topoisomerase activity to support the subsequent replication-related topological stress.

/pdf/bacterial-type-1a-topoisomerases-maintain-the-stability-of-k1xpfle57x.pdf

Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress

Vlachos-Breton E

Papers

Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress