Some new approaches to conventional methods for the study of protein-DNA interactions
Creators: name: Boissy, Robert James
Publisher: name: University of British Columbia
Year: 2010

description: Two important experimental techniques used to study eukaryotic, sequence-specific DNA-binding proteins are electrophoretic mobility-shift assays (EMSAs) and sequence-specific DNA-affinity chromatography. This thesis describes some new approaches to these frequently used methods, which were used to study a DNA-binding protein (ceHSBF) with an apparent specificity for a conserved DNA sequence element (the heat-shock element or HSE) that mediates the transcriptional regulation of heat-shock-inducible genes in the soil nematode Caenorlzabditis elegans. Although C. elegans has attracted extensive research interest, a lack of appropriate and reliable experimental methods has severely limited biochemical studies of sequence-specific DNA-binding proteins from this species. As a first step towards addressing this problem, I developed a simple and effective procedure for isolating C. elegans nuclei. I also developed a new class of EMSA electrophoresis buffers and established the running conditions appropriate for their use. The distinctive electrolyte in these buffers is monopotassium hydrogen glutamate (KHG1u), which provides both a physiological concentration of potassium ions (100- 150 mM) and a counterion species (glutamate) that is well-known for its ability to stabilize protein-DNA interactions. Two protein-DNA complexes that appeared to be specific for an HSE-bearing probe were detected using C elegans nuclear extracts and the KHG1u-based EMSA system. These complexes were believed to have been formed by two distinct types of putative ceHSBF activity, which were designated as ceHSBF 0and ceHSBF. The Rf(-.0. 1-0.2) of the protein-HSE complex formed by the putative ceHSBF 0activity agrees with the known Rf of the complex formed by most of the homotrimeric heat-shock factors from other species. The Rf (—0.4-0.5) of the protein-HSE complex formed by the putative ceHSBFm activity suggests that this protein is either a discrete proteolytic degradation product of ceHSBF 0,or that ceHSBFm is a novel HSE-specific DNAbinding protein. The putative ceHSBFm activity is readily detectable in the presence of moderate concentrations of KC1, nonionic detergents, or a 200,000-fold mass-excess (relative to the labeled HSE probe) of the nonspecific competitor DNA poly(dI-dC).poly(dI-dC), but cannot be detected if the nuclear extract has been exposed to proteinase K or a 50-fold excess of unlabeled HSE probe DNA. In the second part of this thesis I examined various parameters that influence the covalent immobilization of unmodified, double-stranded DNA on acyl hydrazide-derivatized chromatography resins. Commercially available, derivatized agarose or poly(methyl methacrylate) resins were examined. A reaction involving the transainination of transiently single-stranded deoxycytidylate residues by acyl hydrazide groups of the resin is proposed to explain the immobilization. Some noteworthy properties of this immobilization chemistry include satisfactory yields and efficiencies, with 1.0 mg of a 2,686 base-pair fragment of DNA immobilized per millilitre of resin, with 80% efficiency, under standard reaction conditions (6 hours, 30-37 °C, pH 4.20); greater safety and convenience compared to CNBr-activated agarose resins; and the suitability of immobilized DNA for use as an affinity chromatography ligand, or as a solid-phase substrate for various physical, chemical and enzymatic reactions performed at ambient or elevated temperatures.