Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA

James R Portman, Gwen M Brouwer, Jack M Bollins, Nigel J Savery, Terence R Strick*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
30 Downloads (Pure)

Abstract

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.
Original languageEnglish
Article numbere2019630118
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number15
Early online date7 Apr 2021
DOIs
Publication statusPublished - 13 Apr 2021

Bibliographical note

Funding Information:
Horizons 2020 Innovative Training Network “DNAREPAIRMAN.” J.B. was supported by a studentship from the Biotechnology and Biological Sciences Research Council South West Biosciences Doctoral Training Partnership (BBSRC SWBio DTP). J.R.P. acknowledges support from the Frontières du Vivant–Programme Bettencourt doctoral program. Research on this topic in the T.R.S. laboratory was supported by grants from the French Agence Nationale de la Rechrche (ANR-17-CE11-0042) and the Ligue Nationale Contre le Cancer program for Core Research Teams (“Equipes Labellisées”), the NanoRep grant funded by Paris Sciences et Lettres University, as well as core funding from the CNRS, the Ecole Normale Supérieure, and INSERM.

Publisher Copyright:
© This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Keywords

  • transcription-coupled repair
  • R-loops
  • mutagenesis
  • single molecule
  • magnetic trapping
  • Topological domain
  • Supercoiling
  • Mfd
  • Transcription
  • Single-molecule

Fingerprint

Dive into the research topics of 'Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA'. Together they form a unique fingerprint.

Cite this