title: Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand-DNA complexes: the interaction between DNA and calichearubicin B.
abstract: We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (phi) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand-DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin gamma1I. The unwinding angle for CRB calculated by this method is -5. 3 +/- 0.5 degrees. Its Ka value is 0.20 x 10(6) M-1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, 06511, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Zeman
authors.forename: K M
authors.initials: KM
authors.lastname: Depew
authors.forename: S J
authors.initials: SJ
authors.lastname: Danishefsky
authors.forename: D M
authors.initials: DM
authors.lastname: Crothers
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry* / Q000378:metabolism*; D003630:Daunorubicin / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D000617:Aminoglycosides; D004270:DNA, Circular; D007364:Intercalating Agents; D008024:Ligands; C111805:calichearubicin B; D004264:DNA Topoisomerases, Type I; D004996:Ethidium; D003630:Daunorubicin