title: Initiation of transcription-coupled repair characterized at single-molecule resolution.
abstract: Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.
authors.affiliation: Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité F-75205 Paris, France.
authors.forename: Kévin
authors.initials: K
authors.lastname: Howan
authors.forename: Abigail J
authors.initials: AJ
authors.lastname: Smith
authors.forename: Lars F
authors.initials: LF
authors.lastname: Westblade
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joly
authors.forename: Wilfried
authors.initials: W
authors.lastname: Grange
authors.forename: Sylvain
authors.initials: S
authors.lastname: Zorman
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Savery
authors.forename: Terence R
authors.initials: TR
authors.lastname: Strick
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D004249:DNA Damage; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011740:Pyrimidine Dimers / Q000737:chemistry / Q000378:metabolism; D061805:Transcription Elongation, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D061785:Transcription Initiation, Genetic; D061806:Transcription Termination, Genetic; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D011740:Pyrimidine Dimers; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases