Journal
GENES
Volume 9, Issue 4, Pages -Publisher
MDPI
DOI: 10.3390/genes9040190
Keywords
DNA repair; endonuclease III; 5,6-dihydrouracil; stopped-flow enzyme kinetics; thermodynamics; fluorescence
Categories
Funding
- Russian State funded budget project [VI.57.1.2, 0309-2018-0001]
- Russian Foundation for Basic Research [16-04-00037]
- Russian Scientific Foundation [16-14-10038]
- Russian Science Foundation [16-14-10038] Funding Source: Russian Science Foundation
Ask authors/readers for more resources
Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tC degrees), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 degrees C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Aspl38Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available