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.
Highlights
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
We have previously created and validated a chromosomally expressed fusion of Mfd to the yellow fluorescent protein (YPet) that is functional in transcription-coupled repair (TCR), and that is recruited to DNA via paused/stalled ternary elongation complex (TEC) with a bound lifetime of 18 ± 1 s in wild-type cells[21]
We demonstrate that in live bacterial cells dissociation of Mfd requires ATP hydrolysis at the proximal site of UvrA, and a UvrB protein with an intact β-hairpin motif (Fig. 5)
Summary
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-UvrA2UvrB complex or alternate reaction intermediates in cells remains unclear. DNA damage on the transcribed strand is repaired at a rate faster than lesions on the non-transcribed strand[1–3] This enhanced transcription-coupled repair (TCR) is attributed to RNA polymerase (RNAP) acting as a damage sensor, followed by the transcription-repair coupling factor (TRCF)-. In the model organism Escherichia coli, the prokaryotic TRCF, Mfd recognizes stalled transcriptional complexes at sites of lesions[4] and roadblocks[7,8] and orchestrates termination of transcription and recruitment of NER machinery (UvrAB) to the site. This motor domain orchestrates translocation of Mfd in the 3′–5′ direction with respect to the transcribed strand[12–14]
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