Journal
JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS
Volume 35, Issue 5, Pages 950-967Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/07391102.2016.1171800
Keywords
base excision repair; DNA glycosylases; conformational dynamics; enzyme kinetics; NMR; alkyladenine DNA glycosylase
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Funding
- Russian Ministry of Education and Science [SS-7564.2016.4]
- Russian Academy of Sciences, Molecular & Cell Biology Program [6.11]
- Russian Foundation for Basic Research [14-03-00453, 14-04-00531, 15-04-00467, 15-34-20121, 16-04-00037]
- Russian Science Foundation [14-14-00063, 15-13-20035]
- Russian Science Foundation [14-14-00063] Funding Source: Russian Science Foundation
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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 (epsilon A), 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)
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