Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 32198374.
- Also identified by DOI 10.1038/s41467-020-15182-3 and PMC identifier 7083905.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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-UvrA<sub>2</sub>-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-UvrA<sub>2</sub> complex. Further, Mfd-UvrA<sub>2</sub>-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.
Medical subject headings
- DNA Repair
- DNA Repair Enzymes
- Escherichia coli
- Escherichia coli Proteins
- Single Molecule Imaging
- Transcription Factors