G. Eric Plum

Influence of Drug Binding on DNA Hydration: Acoustic and Densimetric Characterizations of Netropsin Binding to the Poly(dAdT).cntdot.Poly(dAdT) and Poly(dA).cntdot.Poly(dT) Duplexes and the Poly(dT).cntdot.Poly(dA).cntdot.Poly(dT) Triplex at 25 .degree.C

We use high-precision acoustic and densimetric techniques to determine, at 25 degrees C, the changes in volume, delta V, and adiabatic compressibility, delta Ks, that accompany the binding of netropsin to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes, as well as to the poly(dT).poly(dA).poly(dT) triplex. We find that netropsin binding to the heteropolymeric poly(dAdT).poly(dAdT) duplex is accompanied by negative changes in volume, delta V, and small positive changes in compressibility, delta Ks. By contrast, netropsin binding to the homopolymeric poly(dA).poly(dT) duplex is accompanied by large positive changes in both volume, delta V, and compressibility, delta Ks. Furthermore, netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes changes in both volume and compressibility that are nearly twice as large as those observed when netropsin binds to the poly(dA).poly(dT) duplex. We interpret these macroscopic data in terms of binding-induced microscopic changes in the hydration of the DNA structures and the drug. Specifically, we find that netropsin binding induces the release of approximately 22 waters from the hydration shell of the poly(dAdT).poly(dAdT) heteropolymeric duplex, approximately 40 waters from the hydration shell of the poly(dA).poly(dT) homopolymeric duplex, and about 53 waters from the hydration shell of the poly(dA).poly(dT), induces the release of 18 more water molecules than netropsin binding to the heteropolymeric duplex, poly(dAdT).poly(dAdT). On the basis of apparent molar volume, phi V, and apparent molar adiabatic compressibility, phi Ks, values for the initial drug-free and final drug-bound states of the two all-AT duplexes, we propose that the larger dehydration of the poly(dA).poly(dT) duplex reflects, in part, the formation of a less hydrated poly(dA).poly(dT)-netropsin complex compared with the corresponding poly(dAdT).poly(dAdT)-netropsin complex. In conjunction with our previously published entropy data [Marky, L. A., & Breslauer, K. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4359-4363], we calculate that each water of hydration released to the bulk solvent by ligand binding contributes 1.6 cal K-1 mol-1 to the entropy of binding. This value corresponds to the average difference between the partial molar entropy of water in the bulk state and water in the hydration shells of the two all-AT duplexes. When netropsin binds to the poly(dT).poly(dA).poly(dT) triplex, the changes in both volume and compressibility suggest that the binding event induces more dehydration of the triplex than of the duplex state. Specifically, we calculate that netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes the release of 13 more waters than netropsin binding to the poly(dA).poly(dT) duplex.(ABSTRACT TRUNCATED AT 400 WORDS)

The thermodynamics of DNA structures that contain lesions of guanine tetrads

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

Calorimetry of proteins and nucleic acids

The availability of sensitive calorimetric instrumentation has led to a considerable increase in thermodynamic studies of proteins, nucleic acids, and their interactions. This article reviews some of the recent contributions of calorimetry to characterizing the thermodynamic origins of protein and nucleic acid stability and conformational preferences, as well as the interactions of proteins with each other, with small molecules, and with nucleic acids.

Energetics of Lesion Recognition by a DNA Repair Protein: Thermodynamic Characterization of Formamidopyrimidine-glycosylase (Fpg) Interactions with Damaged DNA Duplexes

As part of an overall effort to map the energetic landscape of the base excision repair pathway, we report the first thermodynamic characterization of repair enzyme binding to lesion-containing duplexes. Isothermal titration calorimetry (ITC) in conjunction with spectroscopic measurements and protease protection assays have been employed to characterize the binding of Escherichia coli formamidopyrimidine-glycosylase (Fpg), a bifunctional repair enzyme, to a series of 13-mer DNA duplexes. To resolve energetically the binding and the catalytic events, several of these duplexes are constructed with non-hydrolyzable lesion analogs that mimic the natural 8-oxo-dG substrate and the abasic-like intermediates. Specifically, one of the duplexes contains a central, non-hydrolyzable, tetrahydrofuran (THF) abasic site analog, while another duplex contains a central, carbocyclic substrate analog (carba-8-oxo-dG). ITC-binding studies conducted between 5.0 degrees C and 15.0 degrees C reveal that Fpg association with the THF-containing duplex is characterized by binding free energies that are relatively invariant to temperature (deltaG approximately -9.5 kcalmol(-1)), in contrast to both the reaction enthalpy and entropy that are strongly temperature-dependent. Complex formation between Fpg and the THF-containing duplex at 15 degrees C exhibits an unfavorable association enthalpy (deltaH=+7.5 kcalmol(-1)) that is compensated by a favorable association entropy (TdeltaS=+17.0 kcalmol(-1)). The entropic nature of the binding interaction, coupled with the large negative heat capacity (deltaC(p)=-0.67 kcaldeg(-1)mol(-1)), is consistent with Fpg complexation to the THF-containing duplex involving significant burial of non-polar surface areas. By contrast, under the high ionic strength buffer conditions employed herein (200 mM NaCl), no appreciable Fpg affinity for the carba-8-oxo-dG substrate analog is detected. Our results suggest that initial Fpg recognition of a damaged DNA site is predominantly electrostatic in nature, and does not involve large contact interfaces. Subsequent base excision presumably facilitates accommodation of the resulting lesion site into the binding pocket, as the enzyme interaction with the THF-containing duplex is characterized by high affinity and a large negative heat capacity change. Our data are consistent with a pathway in which Fpg glycosylase activity renders the base excision product a preferred ligand relative to the natural substrate, thereby ensuring the fidelity of removing highly reactive and potentially mutagenic abasic-like intermediates through catalytic elimination reactions.

The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies

We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between –9.8 and –16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template–primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.

DNA repair and DNA triplet repeat expansion: the impact of abasic lesions on triplet repeat DNA energetics.

Enhanced levels of DNA triplet expansion are observed when base excision repair (BER) of oxidative DNA base damage (e.g., 8-oxo-dG) occurs at or near CAG repeat sequences. This observation suggests an interplay between processing mechanisms required for DNA repair and expansion pathways that yield genotypes associated with many neurological/developmental disorders. It has been proposed that DNA expansion involves the transient formation within the triplet repeat domains of non-native slipped DNA structures that are incorrectly processed by the BER machinery of repair during DNA synthesis. We show here that replacement within a triplet repeat bulge loop domain of a guanosine residue by an abasic site, the universal BER intermediate, increases the population of slipped/looped DNA structures relative to the corresponding lesion-free construct. Such abasic lesion-induced energetic enhancement of slipped/looped structures provides a linkage between BER and DNA expansion. We discuss how the BER machinery of repair may be influenced by abasic-induced energetic alterations in the properties of regions proximal to and/or within triplet repeat domains, thereby potentially modulating levels of DNA expansion.

Energy Landscapes of Dynamic Ensembles of Rolling Triplet Repeat Bulge Loops: Implications for DNA Expansion Associated with Disease States

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as “rollamers”. Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain (“rollamerization”). We demonstrate that such “rollameric” migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor “rollamers” with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic “universal hinge” relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.

Energy crosstalk between DNA lesions: implications for allosteric coupling of DNA repair and triplet repeat expansion pathways.

Energy coupling between distal DNA domains may have profound regulatory consequences for biological processes, allowing for allosteric control of nucleic acid function. Repair of oxidative lesions at or near triplet repeat domains can enhance DNA expansion events that result in debilitating disease states. We report here position, distance, and lesion-dependent energy crosstalk between pairs of lesions in a triplet repeat bulge loop and an adjacent duplex domain. We discuss the implications of such coupled communication between lesions in distal loop and duplex domains for lesion repair and DNA expansion associated with diseases.

Energetic coupling between clustered lesions modulated by intervening triplet repeat bulge loops: Allosteric implications for DNA repair and triplet repeat expansion

Clusters of closely spaced oxidative DNA lesions present challenges to the cellular repair machinery. When located in opposing strands, base excision repair (BER) of such lesions can lead to double strand DNA breaks (DSB). Activation of BER and DSB repair pathways has been implicated in inducing enhanced expansion of triplet repeat sequences. We show here that energy coupling between distal lesions (8oxodG and/or abasic sites) in opposing DNA strands can be modulated by a triplet repeat bulge loop located between the lesion sites. We find this modulation to be dependent on the identity of the lesions (8oxodG vs. abasic site) and the positions of the lesions (upstream vs. downstream) relative to the intervening bulge loop domain. We discuss how such bulge loop-mediated lesion crosstalk might influence repair processes, while favoring DNA expansion, the genotype of triplet repeat diseases.

UNIT 7.3 Optical Methods

Equilibrium optical melting curves are used in this unit to determine thermodynamic parameters of nucleic acid complex formation. This section contains all of the relevant equations and a discussion of which are most appropriate to a given situation. Additionally, procedures are given for making preliminary determinations of molar extinction coefficients and for determining the number of oligonucleotides in a complex. The section on extinction coefficients is particularly essential to anyone needing to know solution concentrations.