Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

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Abstract

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

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Author and article information

Contributors

Sarah S Henrikus

Camille Henry

Amy E McGrath:

ORCID: http://orcid.org/0000-0001-9408-4521

Slobodan Jergic

John P McDonald

Yvonne Hellmich

Steven T Bruckbauer

Matthew L Ritger

Megan E Cherry

Elizabeth A Wood

Phuong T Pham

Myron F Goodman

Roger Woodgate:

ORCID: http://orcid.org/0000-0001-5581-4616

Michael M Cox:

ORCID: http://orcid.org/0000-0003-3606-5722

Antoine M van Oijen

Harshad Ghodke

Andrew Robinson:

ORCID: http://orcid.org/0000-0002-3544-0976

Journal

Journal ID (nlm-ta): Nucleic Acids Res

Journal ID (iso-abbrev): Nucleic Acids Res

Journal ID (publisher-id): nar

Title: Nucleic Acids Research

Publisher: Oxford University Press

ISSN (Print): 0305-1048

ISSN (Electronic): 1362-4962

Publication date (Print): 04 September 2020

Publication date (Electronic): 20 July 2020

Publication date PMC-release: 20 July 2020

Volume: 48

Issue: 15

Pages: 8490-8508

Affiliations

Molecular Horizons Institute and School of Chemistry and Molecular Bioscience, University of Wollongong , Wollongong, NSW 2522, Australia

Illawarra Health and Medical Research Institute , Wollongong, NSW 2522, Australia

Department of Biochemistry, University of Wisconsin-Madison , WI 53706, USA

Molecular Horizons Institute and School of Chemistry and Molecular Bioscience, University of Wollongong , Wollongong, NSW 2522, Australia

Illawarra Health and Medical Research Institute , Wollongong, NSW 2522, Australia

Molecular Horizons Institute and School of Chemistry and Molecular Bioscience, University of Wollongong , Wollongong, NSW 2522, Australia

Illawarra Health and Medical Research Institute , Wollongong, NSW 2522, Australia

Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, MD 20892, USA

Institute of Biochemistry, Goethe Universität , Frankfurt 3MR4+W2, Germany

Department of Biochemistry, University of Wisconsin-Madison , WI 53706, USA

Molecular Horizons Institute and School of Chemistry and Molecular Bioscience, University of Wollongong , Wollongong, NSW 2522, Australia

Illawarra Health and Medical Research Institute , Wollongong, NSW 2522, Australia

Department of Biochemistry, University of Wisconsin-Madison , WI 53706, USA

Department of Biological Sciences, University of Southern California , Los Angeles, CA 90089, USA

Departments of Biological Sciences and Chemistry, University of Southern California , Los Angeles, CA 90089, USA