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

doi:10.1073/pnas.2019630118

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 journal › Article (Academic Journal) › peer-review

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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 language	English
Article number	e2019630118
Number of pages	7
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	118
Issue number	15
Early online date	7 Apr 2021
DOIs	https://doi.org/10.1073/pnas.2019630118
Publication status	Published - 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

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title = "Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA",

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.",

keywords = "transcription-coupled repair, R-loops, mutagenesis, single molecule, magnetic trapping, Topological domain, Supercoiling, Mfd, Transcription, Single-molecule",

author = "Portman, {James R} and Brouwer, {Gwen M} and Bollins, {Jack M} and Savery, {Nigel J} and Strick, {Terence R}",

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 Fronti{\`e}res 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 Labellis{\'e}es”), the NanoRep grant funded by Paris Sciences et Lettres University, as well as core funding from the CNRS, the Ecole Normale Sup{\'e}rieure, and INSERM. Publisher Copyright: {\textcopyright} This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).",

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T1 - Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA

AU - Portman, James R

AU - Brouwer, Gwen M

AU - Bollins, Jack M

AU - Savery, Nigel J

AU - Strick, Terence R

N1 - 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).

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Y1 - 2021/4/13

N2 - 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.

AB - 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.

KW - transcription-coupled repair

KW - R-loops

KW - mutagenesis

KW - single molecule

KW - magnetic trapping

KW - Topological domain

KW - Supercoiling

KW - Mfd

KW - Transcription

KW - Single-molecule

U2 - 10.1073/pnas.2019630118

DO - 10.1073/pnas.2019630118

M3 - Article (Academic Journal)

C2 - 33827922

SN - 0027-8424

VL - 118

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 15

M1 - e2019630118

ER -

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

Abstract

Bibliographical note

Keywords

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