TY - JOUR

T1 - The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

AU - Kuznetsov, N. A.

AU - Kiryutin, A. S.

AU - Kuznetsova, A. A.

AU - Panov, M. S.

AU - Barsukova, M. O.

AU - Yurkovskaya, A. V.

AU - Fedorova, O. S.

N1 - Publisher Copyright: © 2016 Informa UK Limited, trading as Taylor & Francis Group.

PY - 2017/4/4

Y1 - 2017/4/4

N2 - 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, Tm, 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, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(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.

AB - 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, Tm, 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, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(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.

KW - alkyladenine DNA glycosylase

KW - base excision repair

KW - conformational dynamics

KW - DNA glycosylases

KW - enzyme kinetics

KW - NMR

KW - Oligodeoxyribonucleotides

KW - Humans

KW - Substrate Specificity

KW - DNA Glycosylases/chemistry

KW - Thermodynamics

KW - DNA/chemistry

KW - DNA Repair

KW - Nuclear Magnetic Resonance, Biomolecular

KW - Protein Binding

KW - Catalysis

KW - Kinetics

KW - Transition Temperature

KW - STEADY-STATE KINETICS

KW - ESCHERICHIA-COLI

KW - SACCHAROMYCES-CEREVISIAE

KW - CONFORMATIONAL DYNAMICS

KW - NUCLEIC-ACIDS

KW - HUMAN 8-OXOGUANINE-DNA GLYCOSYLASE

KW - ALKYLATING-AGENTS

KW - BASE-EXCISION

KW - N-GLYCOSYLASE

KW - HYDROGEN-EXCHANGE

UR - http://www.scopus.com/inward/record.url?scp=84963864305&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/65f38e6b-1aa4-3f3b-9f84-0e0675f82029/

U2 - 10.1080/07391102.2016.1171800

DO - 10.1080/07391102.2016.1171800

M3 - Article

C2 - 27025273

AN - SCOPUS:84963864305

VL - 35

SP - 950

EP - 967

JO - Journal of Biomolecular Structure and Dynamics

JF - Journal of Biomolecular Structure and Dynamics

SN - 0739-1102

IS - 5

ER -