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Anna Seck

Nucleotide Excision Repair: New approaches to investigate its exceptionally broad substrate specificity

Published on 19 January 2021
Thesis presented January 19, 2021

Abstract:
The Nucleotide Excision Repair (NER) pathway is responsible for the removal of a wide range of structurally and chemically variable bulky lesions causing minor to important distortions to the DNA helix. These lesions can be introduced by exogenous factors like UV-irradiation or diverse carcinogens. How such a diverse set of lesions are recognized and removed by just a small set of proteins, the Uvr proteins in bacteria, remains largely misunderstood. To address this question, we have developed an in vitro incision assay using the NER system from the radiation resistant bacterium, Deinococcus radiodurans, composed of 4 proteins: UvrA1, UvrB, UvrC and the DNA helicase UvrD, which is dispensable for the incision activity. In contrast to earlier studies that have made use of Uvr proteins from thermophilic bacteria, the reconstitution of the UvrABC system relied on the use of purified proteins from a single, mesophilic bacterium. The incision activity was evaluated on either short DNA oligonucleotides containing modified bases or on plasmid DNA treated with a number of damaging agents.
In this study, we have optimized the incision assay and determined the minimal components and their optimal concentrations needed for efficient repair of bulky lesions. This assay has enabled us to explore the substrate specificity of bacterial NER notably by determining the nature and abundance of lesions repaired by the Uvr proteins by HPLC-MS/MS, the kinetics of repair and the nature of the released product resulting from the dual cleavage reaction. Our data reveal clear differences in the extent of processing of different substrates. The incision assay was also used to decipher the role of the different domains of UvrC in the repair process, which remains one of the most enigmatic steps of NER. Overall, these studies have shed light on several aspects of this repair pathway and provide the scientific community with a powerful tool for future studies.

Keywords:
UvrABC, Nucleotide Excision Repair, DNA lesions, Deinococcus radiodurans, damaging agents, HPLC-MS/MS.