The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase Full article

Journal

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102

Output data

Year: 2017, Volume: 35, Number: 5, Pages: 950-967 Pages count : 18 DOI: 10.1080/07391102.2016.1171800

Tags

base excision repair, DNA glycosylases, conformational dynamics, enzyme kinetics, NMR, alkyl,adenine DNA glycosylase

Authors

Kuznetsov N.A. ^1,3 , Kiryutin A.S. ^2,3 , Kuznetsova A.A. ¹ , Panov M.S. ^2,3 , Barsukova M.O. ³ , Yurkovskaya A.V. ^2,3 , Fedorova O.S. ¹

Affiliations

1	Institute of Chemical Biology and Fundamental Medicine, Lavrentyev Ave. 8, Novosibirsk 630090, Russia
2	International Tomography Center SB RAS, Institutskaya 3a, Novosibirsk 630090, Russia
3	Department of Natural Sciences, Novosibirsk State University, Pirogova St. 2, Novosibirsk 630090, Russia

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T m, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T m(F/T) < T m(εA/T) < T m(Hx/T) < T m(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Kuznetsov N.A. , Kiryutin A.S. , Kuznetsova A.A. , Panov M.S. , Barsukova M.O. , Yurkovskaya A.V. , Fedorova O.S.
The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase
Journal of Biomolecular Structure and Dynamics. 2017. V.35. N5. P.950-967. DOI: 10.1080/07391102.2016.1171800 WOS Scopus

Submitted:	Dec 8, 2015
Accepted:	Mar 24, 2016
Published online:	Apr 19, 2016

Web of science:	WOS:000399482000003
Scopus:	2-s2.0-84963864305

DB	Citing
Scopus	12
Web of science	12