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.
Highlights
Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life
After damage recognition, which requires ATP binding to the distal site, ATP hydrolysis in the proximal site is necessary for UvrB recruitment to DNA
We propose that ATP hydrolysis in the distal site facilitates release of UvrA from the repair complex, allowing UvrB to further verify the presence of damage and coordinate the downstream steps of NER
Summary
Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. According to currently accepted models, dimeric UvrA forms a stable complex with either one or two UvrB molecules[3,4,5,10,11] This UvrA–UvrB damage sensor scans the genome to locate DNA lesions. The UvrA dimer belongs to the ATP-binding cassette family of ATPases[12], but unlike the majority of the family members, UvrA has two ATP-binding sites per monomer, designated ‘proximal’ and ‘distal’[6,7,8] The activity of both sites is required for NER, and it was demonstrated that both sites work co-operatively[13]. We propose that ATP hydrolysis in the distal site facilitates release of UvrA from the repair complex, allowing UvrB to further verify the presence of damage and coordinate the downstream steps of NER
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