Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B (DNA unwindingydrug binding constantsydaunorubicinycalicheamicinyethidium bromide)

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 DNAyligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (f) 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 g1 . The unwinding angle for CRB calculated by this method is 25.3 6 0.5°. Its Ka value is 0.20 3 106 M21. 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. By now there is little doubt that intercalation is a phenomenon of widespread biological importance. The intercalation model (1) describes a lengthening of the DNA helix and a concomitant unwinding of the phosphodiester backbone to accommodate a molecule of intercalator. As this unwinding varies with intercalator, the unwinding angle (f) is a valuable biophysical parameter when evaluating the ability of a compound to interact with DNA. It represents the number of degrees by which the DNA is unwound about its helical axis per ligand molecule bound. To date, most experimental approaches for measuring f have exploited the special conformational characteristics of closed circular DNA (ccDNA). As isolated from prokaryotes and viruses, ccDNA is negatively supercoiled, that is to say, its writhe (Wr), linking difference (DLk), and superhelical density (s, the average number of superhelical turns per 10 bp) are negative.§ Upon binding by intercalator, ccDNA is unwound so that DLk and s each become less negative (Fig. 1B). For a certain critical amount of bound intercalator, all superhelicity disappears and the ccDNA exists as a relaxed circle in which the helical axis lies f lat in a plane. For this example, DLk, Wr, and s equal zero (Fig. 1C). Further titration with intercalator past this topological equivalence point continues to unwind the helix, introducing supercoils in an opposite sense so that both DLk and s become positive (Fig. 1D). Early hydrodynamic methods of studying s and f were based on changes in the sedimentation velocity (4–8), buoyant density (9–11), and viscosity (8) of ccDNA when bound by intercalator. The discovery of the enzyme topoisomerase I (Topo I) (12, 13) permitted other experimental approaches to both f and s, which were extended to studies of complexes between DNA and such topology-perturbing proteins as EcoRI (14), Xenopus transcription factor A (15), RNA polymerase (16–18), and the lac repressor (19). Topo I facilitates the thermodynamic equilibration between supercoiled and relaxed forms of ccDNA by creating a transient single-stranded nick at which superhelical strain can be relieved. Methods for the determination of f and s that have used Topo I and intercalating ligand are based on the processes shown in Fig. 1. When ccDNA that has been intercalated to some extent is acted on by Topo I, a fully relaxed species is generated whose twist (Tw) and Wr reflect the presence of bound intercalator. In this fully relaxed species, Wr 5 DLk 5 0. When this intercalator is removed, the sudden change in Tw is partially offset by a compensatory Wr so that Lk, an invariant topological property intrinsic to any intact molecule of ccDNA, remains unchanged. This change in Wr manifests itself conformationally as a reappearance of superhelicity, the magnitude of which is determined by the original amount of intercalator bound at the time of enzymatic religation. Quantitating this superhelicity in some way enables a calculation of f or s. In reality, the resulting ccDNA is not homogeneous and actually exists as a family of topoisomers differing by integral increments of DLk. Thermal conformational f luctuations at the time of ccDNA religation give rise to a Boltzman distribution of topoisomers around an average DLk (20–23). This central DLk (DLkc) is taken as the average most abundant The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y954327-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ccDNA, closed circular DNA; Topo I, topoisomerase I; CRB, calichearubicin B; DA, daunorubicin; CLM, calicheamicin g1 I ; EB, ethidium bromide; Wr, writhe; DLk, linking difference; Tw, twist; 1D, one dimensional; MGOS, methyl glycoside of the CLM oligosaccharide. A commentary on this article begins on page 4092. ‡To whom reprint requests should be addressed. §Lk 5 Tw 1 Wr, where Tw is the twist about the helical axis and Wr is the writhe of the helical axis through three-dimensional space. Whereas Tw and Wr are each geometric properties that vary with environmental conditions such as temperature and ionic strength, Lk is a topological property that is invariant in the absence of singlestranded nicks. DLk is defined as the difference in superhelical turns between a fully relaxed species for which Wr 5 0 and species j of nonzero Wr. Although DLk can assume a fractional value, Lk is integral by definition. Excellent treatments of ccDNA topology exist in the literature (2, 3).