Biomedical subjects

Kenneth J Breslauer

Publications and source records attributed to Kenneth J Breslauer.

5 recordsLinked to original sources

PubMed2006

What drives proteins into the major or minor grooves of DNA?

The energetic profiles of a significant number of protein-DNA systems at 20 degrees C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy-driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves; namely, that the water in the A+T-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic.

Base Composition↗

PubMed2005

Communication between noncontacting macromolecules.

Molecular interactions are the language that molecules use to communicate recognition, binding, and regulation, events central to biological control mechanisms. Traditionally, such interactions involve direct, atom-to-atom, noncovalent contacts, or indirect contacts bridged by relatively fixed solvent molecules. Here we discuss a third class of molecular communication that, to date, has received less experimental attention, namely solvent-mediated communication between noncontacting macromolecules. This form of communication can be understood in terms of fundamental, well-established principles (coupled equilibria and linkage thermodynamics) that govern interactions between individual polymers and their solutions. In contrast to simple solutions used in laboratory studies, biological systems contain a multitude of nominally noninteracting biopolymers within the same solution environment. The exquisite control of biological function requires some form of communication between many of these solution components, even in the absence of direct and/or indirect contacts. Such communication must be considered when describing potential mechanisms of biological regulation.

Biophysics↗

PubMed2004

Conformational screening of oligonucleotides by variable-temperature high performance liquid chromatography: dissecting the duplex-hairpin-coil equilibria of d(CGCGAATTCGCG).

This study probes the potential of variable-temperature high performance liquid chromatography (VT-HPLC) as a tool for dissecting and modulating nucleic acid structural transitions, using as a model the duplex-hairpin-coil transitions of d(CGCGAATTCGCG). It is demonstrated that VT-HPLC, combined with diode-array detection of the uv signal, enables, for the first time, a physical separation of spectroscopically distinct species that can be assigned to the duplex, hairpin, and coil forms of d(CGCGAATTCGCG). Although the species are spectroscopically distinguishable, they are not readily isolated. Hence, if fractions from the peaks for hairpin or duplex forms are collected and subsequently reinjected onto the cartridge, reequilibration occurs, and both hairpin and duplex peaks are observed. Area integration of the peaks corresponding to duplex and hairpin species provides a means to monitor the duplex to hairpin transition at effective concentrations in the nanomolar range, well below that accessible by conventional spectrophotometers. Concentration-dependent equilibrium constants, melting temperatures, and standard state enthalpies extracted from our measurements compare very well with previous literature results, and with our own results that take into account the effect of our solvent conditions [100 mM TEAA (triethylammonium acetate) and variable acetonitrile] on the melting behavior. By combining precise temperature control with separation based on size, physical behavior, and interaction free energies, VT-HPLC provides a powerful tool for both the modulation and the separation of nucleic acid conformations.

Chromatography, High Pressure Liquid↗

PubMed2003

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.

Base Sequence↗

PubMed2003

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.

Base Sequence↗