A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision.
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.
Minetti
Conceição A S A
CA
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854, USA.
Remeta
David P
DP
Breslauer
Kenneth J
KJ
D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D015203:Reproducibility of Results; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D004952:Esters; D009711:Nucleotides; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase