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

C. A S A Minetti, David P. Remeta, Kenneth J. Breslauer

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Abstract

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 ↔ ES) that is enthalpy-driven (ΔHb = -16.4 kcal·mol -1). The activation barrier (ΔG) of 19.6 kcal·mol-1 for the chemical step (ES ↔ P) also is enthalpic in nature (ΔH = 19.2 kcal·mol -1). Formation of the transition state complex from the reactants (E + S ↔ ES), a pathway that reflects Fpg catalytic specificity (kcat/Km) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (ΔGT) 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.

LanguageEnglish (US)
Pages70-75
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number1
DOIs
StatePublished - Jan 8 2008

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Thermodynamics
DNA
Enzymes
Cats
Nucleic Acids
Temperature
8-oxo-7-hydrodeoxyguanosine

All Science Journal Classification (ASJC) codes

  • General
  • Genetics

Cite this

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title = "A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision",
abstract = "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 ↔ ES) that is enthalpy-driven (ΔHb = -16.4 kcal·mol -1). The activation barrier (ΔG‡) of 19.6 kcal·mol-1 for the chemical step (ES ↔ P) also is enthalpic in nature (ΔH‡ = 19.2 kcal·mol -1). Formation of the transition state complex from the reactants (E + S ↔ ES‡), a pathway that reflects Fpg catalytic specificity (kcat/Km) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (ΔGT‡) 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.",
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