Kinetics and thermodynamics of DNA processing by wild type DNA-glycosylase endo III and its catalytically inactive mutant forms

Olga A. Kladova, Lev N. Krasnoperov, Nikita A. Kuznetsov, Olga S. Fedorova

    Research output: Contribution to journalArticlepeer-review

    9 Citations (Scopus)

    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 (5–37ºC). 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.

    Original languageEnglish
    Article number190
    Number of pages18
    JournalGenes
    Volume9
    Issue number4
    DOIs
    Publication statusPublished - 30 Mar 2018

    Keywords

    • 5, 6-dihydrouracil
    • DNA repair
    • Endonuclease III
    • Fluorescence
    • Stopped-flow enzyme kinetics
    • Thermodynamics
    • DAMAGED DNA
    • 5,6-dihydrouracil
    • REPAIR ENZYME
    • BIOLOGICAL CONSEQUENCES
    • ESCHERICHIA-COLI
    • thermodynamics
    • stopped-flow enzyme kinetics
    • CONFORMATIONAL DYNAMICS
    • endonuclease III
    • COLI ENDONUCLEASE-III
    • FREE-RADICALS
    • INTRAHELICAL LESION
    • SUBSTRATE-SPECIFICITY
    • fluorescence
    • LESION RECOGNITION

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