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 enzymecatalyzed, 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 K m of 46.6 nM for the E⅐S 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 (⌬G b) of ؊10.0 kcal⅐mol ؊1 for the binding step (E ؉ S 7 ES) that is enthalpy-driven (⌬H b ‫؍‬ ؊16.4 kcal⅐mol ؊1 ). The activation barrier (⌬G ‡ ) of 19.6 kcal⅐mol ؊1 for the chemical step (ES 7 P) also is enthalpic in nature (⌬H ‡ ‫؍‬ 19.2 kcal⅐mol ؊1 ). Formation of the transition state complex from the reactants (E ؉ S 7 ES ‡ ), a pathway that reflects Fpg catalytic specificity (k cat/Km) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (⌬G T ‡ ) of 9.6 kcal⅐mol ؊1 . These parameters characterize the driving forces that dictate Fpg enzyme efficiency and specificity and elucidate the energy landscape for lesion recognition and repair.8-oxoguanosine ͉ activation energy ͉ energetics ͉ glycosylase/lyase T he number of DNA lesions implicated in genetic diseases and degenerative cell disorders has increased over the past decades (1, 2). Such findings have stimulated efforts to characterize specific enzyme families that function in the recognition and repair of DNA damage (3-7). The majority of existing methods used to determine kinetic parameters for base/nucleotide excision repair (BER/NER) enzymes employ labeling and quenching techniques that measure either product formation or substrate disappearance (3,(8)(9)(10)(11). Although of great value, many of these assays require chemical modifications and/or sampling of the enzyme-catalyzed reaction at discrete time intervals. These features may potentially compromise successful monitoring of intrinsic system properties. To address this challenge, we have developed a continuous hyperchromicity assay (CHA) that exploits the intrinsic free-energy differences of a damaged DNA duplex substrate and the resultant processed product. The method requires minimal sample manipulation and provides a real-time, continuous optical signal to quantify product formation. Because the presence of a damaged base or lesion often exerts profound effects on DNA duplex energetics and stability (12), the solution conditions of the assay are optimized to en...

Fluorescent probes for the analysis of DNA strand scission in base excision repair

“…The principle is that the fluorophore-labeled 5′ fragment of our probe is short enough to dissociate from the quencher strand at the incubation temperature, upon enzymatic cleavage. Destabilization of the duplex upon enzymatic strand scission was used previously to analyze the reaction of a bifunctional enzyme, formamidopyrimidine glycosylase, but the reaction was monitored by hyperchromicity (11).

Figure 2.Structures of the oligonucleotide probes ( A ) and the damaged bases ( B ) used in this study.

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Impact of bistrand abasic sites and proximate orientation on DNA global structure and duplex energetics

“…The overall interest in characterizing DNA duplex damage involving base substitution, insertion, or deletion mutations dates over three decades . During this timeframe, numerous investigations have explored the biochemical, biophysical, and structural impact of chemically modified bases, thereby providing insight regarding the molecular basis of lesion‐induced mutagenesis and cytotoxicity . A unique outcome of ionizing radiation and radiomimetic anticancer treatments is the induction of clustered damage in which two or more lesions are formed within a single DNA helical turn .…”

“…The experimental framework employs a standard reference duplex that has been explored in previous studies to characterize the energetic impact of single base adducts, bulges, and lesions . These damaged bases are site‐specifically introduced within a tridecamer deoxyoligonucleotide duplex comprised of a defined core sequence designed to adopt a non‐self‐complementary bimolecular B‐DNA structure .…”

“…The latter has been widely employed as a suitable site‐specific abasic analog in biophysical studies . Moreover, duplexes harboring this synthetic abasic nucleoside bind repair enzymes (e.g., Fpg) with high affinity, are substrates for APE endonucleases, and inhibit glycosylase‐mediated 8‐oxodeoxyguanosine base excision . A broader goal is to correlate the energetic data with biochemical and structural studies.…”

Microcalorimetry of Macromolecules: The Physical Basis of Biological Structures

“…We are now focusing attention on unraveling intrinsic DNA sequence-specific thermodynamic properties that govern protein binding (as reviewed in [46]). A natural extension of these studies is to evaluate the impact of endogenous/exogenous damaging agents on DNA structure, energetics, and recognition by specialized enzyme repair machineries [49][50][51][52][53][54][55][56]. In conjunction with our concerted effort to elucidate the energetic basis of helix to single strand transitions [28], the realization that DNA dissociation processes are accompanied by a defined heat capacity increment [29,30] has prompted us to develop a comprehensive experimental strategy that characterizes duplex energetics as a function of sequence context and nearest-neighbor contributions.…”