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

Journal Article


Abstract


  • Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair 1–3 . During this reaction, stalled transcription elongation complexes at sites of lesions serve as a signal to trigger the assembly of nucleotide excision repair factors (reviewed in ref. 4,5 ). In the model organism Escherichia coli , the transcription-repair coupling factor Mfd displaces the stalled RNA polymerase and hands-off the stall site to the nucleotide excision repair factors UvrAB for damage detection 6–9 . Despite in vitro evidence, it remains unclear how in live cells the stall site is faithfully handed over to UvrB from RNA polymerase and whether this handoff occurs via the Mfd-UvrA 2 -UvrB complex or via alternate reaction intermediates. Here, we visualise Mfd, the central player of transcription-coupled repair in actively growing cells and determine the catalytic requirements for faithful completion of the handoff during transcription-coupled repair. We find that the Mfd-UvrA 2 complex is arrested on DNA in the absence of UvrB. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition, were also impaired in successful handoff. Our observations demonstrate that in live cells, 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 during transcription-coupled repair.

Publication Date


  • 2019

Citation


  • Ho, H. N., Oijen, A. V., & Ghodke, H. (2019). Single-molecule imaging reveals molecular coupling between transcription and DNA repair in live cells. doi:10.1101/515536

Web Of Science Accession Number


Abstract


  • Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair 1–3 . During this reaction, stalled transcription elongation complexes at sites of lesions serve as a signal to trigger the assembly of nucleotide excision repair factors (reviewed in ref. 4,5 ). In the model organism Escherichia coli , the transcription-repair coupling factor Mfd displaces the stalled RNA polymerase and hands-off the stall site to the nucleotide excision repair factors UvrAB for damage detection 6–9 . Despite in vitro evidence, it remains unclear how in live cells the stall site is faithfully handed over to UvrB from RNA polymerase and whether this handoff occurs via the Mfd-UvrA 2 -UvrB complex or via alternate reaction intermediates. Here, we visualise Mfd, the central player of transcription-coupled repair in actively growing cells and determine the catalytic requirements for faithful completion of the handoff during transcription-coupled repair. We find that the Mfd-UvrA 2 complex is arrested on DNA in the absence of UvrB. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition, were also impaired in successful handoff. Our observations demonstrate that in live cells, 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 during transcription-coupled repair.

Publication Date


  • 2019

Citation


  • Ho, H. N., Oijen, A. V., & Ghodke, H. (2019). Single-molecule imaging reveals molecular coupling between transcription and DNA repair in live cells. doi:10.1101/515536

Web Of Science Accession Number