Nucleotide excision repair (NER) is a conserved and versatile DNA repair pathway found in all domains of life that is responsible for eliminating a wide diversity of DNA lesions from the genome. These include UV-induced pyrimidine dimers, but also bulky adducts caused by smoking or chemotherapeutic agents. In bacteria, NER is initiated by the UvrA and UvrB proteins that together locate the lesion before recruiting a third factor, the dual endonuclease, UvrC, that cleaves the DNA on either side of the damaged site to release a short DNA fragment containing the lesion. Bacterial NER has been studied for over 40 years, and yet the mechanisms involved in the recruitment and activation of the dual incision activity of UvrC at sites of DNA damage are still only poorly understood.
In the present study, we have used biochemical and biophysical approaches including our recently developed in vitro NER assay relying on the UvrA, UvrB and UvrC proteins from Deinococcus radiodurans (Seck et al., Communications Biology, 2022), to probe the DNA binding, UvrB binding and incision activities of wild-type and mutant constructs of UvrC and have constructed the first complete 3D model of a UvrC protein, assembled using the crystallographic structure of the C-terminal region of UvrC and an AlphaFold model of the N-terminal region. Together, this work reveals unexpected features of UvrC proteins and provides important insight into the mechanism of activation of UvrC during NER. In particular, we show that (i) in the absence of any partner, UvrC resides in an inactive state, which cannot perform unwanted incision reactions that would be highly detrimental to the integrity of the genome, and (ii) UvrC activation requires a major conformational rearrangement, which is likely triggered by the binding of UvrC to the UvrB-DNA pre-incision complex. Moreover, we have identified several special features of D. radiodurans UvrC that make it a particularly robust enzyme capable of remaining active for the repair of massively damaged DNA under conditions of stress.
Seck A*, De Bonis S*, Stelter M, Ökvist M, Senarisoy M, Hayek MR, Le Roy A, Martin L, Saint-Pierre C, Silveira CM, Gasparutto D, Todorovic S, Ravanat JL & Timmins J. Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER. (2023) Nucleic Acids Research. DOI : 10.1093/nar/gkad108. * Joint first authors.