Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli

Mathew Stracy, Marcin Jaciuk, Stephan Uphoff, Achillefs N. Kapanidis, Marcin Nowotny, David J. Sherratt () and Pawel Zawadzki ()
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Mathew Stracy: University of Oxford
Marcin Jaciuk: Laboratory of Protein Structure, International Institute of Molecular and Cell Biology
Stephan Uphoff: University of Oxford
Achillefs N. Kapanidis: Biological Physics Research Group, Clarendon Laboratory, University of Oxford
Marcin Nowotny: Laboratory of Protein Structure, International Institute of Molecular and Cell Biology
David J. Sherratt: University of Oxford
Pawel Zawadzki: University of Oxford

Nature Communications, 2016, vol. 7, issue 1, 1-9

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms12568

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