TY - JOUR

T1 - Kinetics and thermodynamics of DNA processing by wild type DNA-glycosylase endo III and its catalytically inactive mutant forms

AU - Kladova, Olga A.

AU - Krasnoperov, Lev N.

AU - Kuznetsov, Nikita A.

AU - Fedorova, Olga S.

PY - 2018/3/30

Y1 - 2018/3/30

N2 - 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.

AB - 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.

KW - 5, 6-dihydrouracil

KW - DNA repair

KW - Endonuclease III

KW - Fluorescence

KW - Stopped-flow enzyme kinetics

KW - Thermodynamics

KW - DAMAGED DNA

KW - 5,6-dihydrouracil

KW - REPAIR ENZYME

KW - BIOLOGICAL CONSEQUENCES

KW - ESCHERICHIA-COLI

KW - thermodynamics

KW - stopped-flow enzyme kinetics

KW - CONFORMATIONAL DYNAMICS

KW - endonuclease III

KW - COLI ENDONUCLEASE-III

KW - FREE-RADICALS

KW - INTRAHELICAL LESION

KW - SUBSTRATE-SPECIFICITY

KW - fluorescence

KW - LESION RECOGNITION

UR - http://www.scopus.com/inward/record.url?scp=85045110051&partnerID=8YFLogxK

U2 - 10.3390/genes9040190

DO - 10.3390/genes9040190

M3 - Article

C2 - 29601551

AN - SCOPUS:85045110051

VL - 9

JO - Genes

JF - Genes

SN - 2073-4425

IS - 4

M1 - 190

ER -