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Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells

Journal Article


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Abstract


  • The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

Publication Date


  • 2020

Citation


  • Ho, H., van Oijen, A. & Ghodke, H. (2020). Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells. Nature communications, 11 (1), 1478.

Scopus Eid


  • 2-s2.0-85082146178

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2289&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1271

Has Global Citation Frequency


Start Page


  • 1478

Volume


  • 11

Issue


  • 1

Place Of Publication


  • United Kingdom

Abstract


  • The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

Publication Date


  • 2020

Citation


  • Ho, H., van Oijen, A. & Ghodke, H. (2020). Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells. Nature communications, 11 (1), 1478.

Scopus Eid


  • 2-s2.0-85082146178

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2289&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1271

Has Global Citation Frequency


Start Page


  • 1478

Volume


  • 11

Issue


  • 1

Place Of Publication


  • United Kingdom