Nucleotide excision repair (NER) is a DNA repair pathway capable of removing of very diverse DNA lesions such as pyrimidine–pyrimidone (6–4) photoproducts (6-4-PP), cyclobutane pyrimidine dimers (CPD) and a wide range of helix-distorting lesions including DNA adducts. In bacteria, this pathway is carried out by the UvrA, UvrB and Uvrc proteins that act sequentially to release a short oligonucleotide containing the damaged base. In this work, we successfully reconstituted an efficient bacterial NER system using an original doubly labeled oligonucleotide and purified UvrABC proteins from the radiation resistant bacterium Deinococcus radiodurans.
Compared to earlier NER systems, two important changes were indeed implemented. First, we made use of an oligonucleotide labeled with a centered fluorescein conjugated to a thymine, a listed substrate of NER, but also an additional red fluorophore on the 5’ end of the lesion-containing strand. These two labels allowed us to track the various products resulting from the incision by the Uvr proteins. Secondly, the proteins were all purified from one single organism D. radiodurans, a mesophilic bacterium. With these very stable proteins, we were able to monitor the assay on longer time courses and work on each component of our system. These two characteristics enabled us to test many conditions in order to optimize the in vitro incision assay to achieve fully incision of the substrate.
This newly developed incision assay will undoubtedly be a useful tool for further studies of the bacterial NER pathway. The fact that it has been fully optimized will now enable us to dissect the contribution of each component to this repair reaction. Building on this work, we thus hope to shed light on some of the complex mechanisms underlying NER in the near future.
Seck A, De Bonis S, Saint-Pierre C, Gasparutto D, Ravanat JL & Timmins J. In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans. Communications Biology (2022). 5, 127. DOI: 10.1038/s42003-022-03064-x.