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Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

1
Institute of Chemical Biology and Fundamental Medicine (ICBFM), 630090 Novosibirsk, Russia
2
Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia
3
New Jersey Institute of Technology, Department of Chemistry and Environment Sciences, University Heights, Newark, NJ 07102, USA
*
Authors to whom correspondence should be addressed.
Genes 2018, 9(4), 190; https://doi.org/10.3390/genes9040190
Received: 27 February 2018 / Revised: 22 March 2018 / Accepted: 27 March 2018 / Published: 30 March 2018
(This article belongs to the Special Issue Nucleic Acid Dynamics and Structure)
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Abstract

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 (tCO), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °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 Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation. View Full-Text
Keywords: DNA repair; endonuclease III; 5,6-dihydrouracil; stopped-flow enzyme kinetics; thermodynamics; fluorescence DNA repair; endonuclease III; 5,6-dihydrouracil; stopped-flow enzyme kinetics; thermodynamics; fluorescence
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Kladova, O.A.; Krasnoperov, L.N.; Kuznetsov, N.A.; Fedorova, O.S. Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms. Genes 2018, 9, 190.

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