In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans.
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
11 02 2022
11 02 2022
Historique:
received:
09
07
2021
accepted:
18
01
2022
entrez:
12
2
2022
pubmed:
13
2
2022
medline:
5
4
2022
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 35149830
doi: 10.1038/s42003-022-03064-x
pii: 10.1038/s42003-022-03064-x
pmc: PMC8837605
doi:
Substances chimiques
Escherichia coli Proteins
0
Endodeoxyribonucleases
EC 3.1.-
Banques de données
figshare
['10.6084/m9.figshare.17212850']
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
127Informations de copyright
© 2022. The Author(s).
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