continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 1; pp. 70 - 75
• Main Authors: Minetti, Conceição A.S.A, Remeta, David P, Breslauer, Kenneth J
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 08.01.2008; National Acad Sciences
• Subjects: Biochemistry; Biological Sciences; Catalysis; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase - chemistry; DNA-Formamidopyrimidine Glycosylase - metabolism; Enthalpy; Enzyme substrates; Enzymes; Escherichia coli - metabolism; Esters - chemistry; Free energy; Glycosylation; Hydrolysis; Kinetics; Lesions; Models, Chemical; Nucleic Acid Conformation; Nucleotides - chemistry; Reproducibility of Results; Temperature; Temperature dependence; Thermodynamics; Time Factors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0710363105

We report a continuous hyperchromicity assay (CHA) for monitoring and characterizing enzyme activities associated with DNA processing. We use this assay to determine kinetic and thermodynamic parameters for a repair enzyme that targets nucleic acid substrates containing a specific base lesion. This optically based kinetics assay exploits the free-energy differences between a lesion-containing DNA duplex substrate and the enzyme-catalyzed, lesion-excised product, which contains at least one hydrolyzed phosphodiester bond. We apply the assay to the bifunctional formamidopyrimidine glycosylase (Fpg) repair enzyme (E) that recognizes an 8-oxodG lesion within a 13-mer duplex substrate (S). Base excision/elimination yields a gapped duplex product (P) that dissociates to produce the diagnostic hyperchromicity signal. Analysis of the kinetic data at 25°C yields a Km of 46.6 nM for the E·S interaction, and a kcat of 1.65 min⁻¹ for conversion of the ES complex into P. The temperature dependence reveals a free energy (ΔGb) of -10.0 kcal·mol⁻¹ for the binding step (E + S [leftright arrow] ES) that is enthalpy-driven (ΔHb = -16.4 kcal·mol⁻¹). The activation barrier (ΔG[double dagger]) of 19.6 kcal·mol⁻¹ for the chemical step (ES [leftright arrow] P) also is enthalpic in nature (ΔH[double dagger] = 19.2 kcal·mol⁻¹). Formation of the transition state complex from the reactants (E + S [leftright arrow] ES[double dagger]), a pathway that reflects Fpg catalytic specificity (kcat/Km) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (ΔGT[double dagger]) of 9.6 kcal·mol⁻¹. These parameters characterize the driving forces that dictate Fpg enzyme efficiency and specificity and elucidate the energy landscape for lesion recognition and repair.