7 research outputs found
Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli
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 provide, using single-molecule
fluorescence imaging, 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 is dissociated from the lesion complex, allowing UvrB to orchestrate the
downstream NER reactions