Thermodynamics Research Today is a free monthly online journal that collates and summarizes the latest research about Thermodynamics, including details on enthalpy, entropy, energy transitions.

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

Minetti CA, Remeta DP, Breslauer KJ

Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854, USA.

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 degrees C yields a K(m) of 46.6 nM for the E.S interaction, and a k(cat) of 1.65 min(-1) for conversion of the ES complex into P. The temperature dependence reveals a free energy (DeltaG(b)) of -10.0 kcal.mol(-1) for the binding step (E + S <--> ES) that is enthalpy-driven (DeltaH(b) = -16.4 kcal.mol(-1)). The activation barrier (DeltaG) of 19.6 kcal.mol(-1) for the chemical step (ES <--> P) also is enthalpic in nature (DeltaH = 19.2 kcal.mol(-1)). Formation of the transition state complex from the reactants (E + S <--> ES), a pathway that reflects Fpg catalytic specificity (k(cat)/K(m)) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (DeltaG(T)) 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.

Published 9 January 2008 in Proc Natl Acad Sci U S A, 105(1): 70-5.
Full-text of this article is available online (may require subscription).

© 2005-2008 Thermodynamics Research Today. All Rights Reserved.