title: Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli.
abstract: Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the 'proximal' and 'distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.|Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors: Mathew
authors: M
authors: Stracy
authors: Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, 4 Ksiecia Trojdena Street, 02-109 Warsaw, Poland.
authors: Marcin
authors: M
authors: Jaciuk
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors: Stephan
authors: 0000-0002-3579-0888
authors: S
authors: Uphoff
authors: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors: Achillefs N
authors: AN
authors: Kapanidis
authors: Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, 4 Ksiecia Trojdena Street, 02-109 Warsaw, Poland.
authors: Marcin
authors: M
authors: Nowotny
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors: David J
authors: DJ
authors: Sherratt
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors: Pawel
authors: P
authors: Zawadzki
mesh_terms: D000251:Adenosine Triphosphatases / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D006868:Hydrolysis; D008853:Microscopy / Q000379:methods; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D000072760:Single Molecule Imaging / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D000255:Adenosine Triphosphate; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases