The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function.
Adenosine Triphosphatases
/ metabolism
Adenosine Triphosphate
/ metabolism
Amino Acid Sequence
Bacterial Proteins
/ metabolism
DNA Breaks, Double-Stranded
/ radiation effects
DNA Breaks, Single-Stranded
/ radiation effects
DNA Damage
DNA Repair
DNA, Bacterial
/ genetics
Endodeoxyribonucleases
/ metabolism
Hydrolysis
Kinetics
Mutation
Mycobacterium tuberculosis
/ enzymology
Sequence Homology, Amino Acid
Ultraviolet Rays
Mycobacterium tuberculosis
UvrABC excinuclease
UvrC
evolution of catalytic scaffolds
non-Walker-type ATPase
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
13
01
2020
revised:
04
05
2020
accepted:
24
06
2020
pubmed:
1
7
2020
medline:
27
7
2021
entrez:
1
7
2020
Statut:
ppublish
Résumé
To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacterium tuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichia coli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability in vitro. In addition, homology modelling of M. tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.
Substances chimiques
Bacterial Proteins
0
DNA, Bacterial
0
Adenosine Triphosphate
8L70Q75FXE
Endodeoxyribonucleases
EC 3.1.-
Adenosine Triphosphatases
EC 3.6.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1179-1200Informations de copyright
© 2020 Federation of European Biochemical Societies.
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