In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Salvatore De Bonis,Jean-Luc Ravanat,Didier Gasparutto,Joanna Timmins,Anna Seck,Christine Saint-Pierre

doi:10.1038/s42003-022-03064-x

Abstract

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

Highlights

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts
Incision reactions were performed using a 50 mer duplex in which one strand was 5′ end-labeled with a red fluorophore and contained a fluorescein-conjugated thymine (FdT) in position 26 (F26-seq1), a well-established substrate of bacterial NER3,36 (Fig. 1b)
We examined the substrate specificity of the D. radiodurans NER system by evaluating its repair efficiency on three additional substrates, all of which were 50 mer DNA duplexes containing at least one fluorophore for detection and a modified base in position 26 (Supplementary Tables 2, 3)

Summary

Introduction

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. In contrast to earlier work, this assay makes use of a doubly-labeled DNA substrate allowing us to identify the different incision products This assay has enabled us to assess the possible involvement of UvrA2 in NER, to perform kinetics studies of the NER process, to probe the substrate specificity of D. radiodurans NER, and to evaluate the role of divalent cations in the dual-incision reaction. By combining this NER assay with MALDI-ToF mass spectrometry analyses, we unambiguously determined the sites of cleavage by UvrC

Methods

Results

Conclusion