Topoisomerase Poisons Bound at a DNA Junction-like Quadruplex James H. Thorpe, Jeanette R. Hobbs, Susana C. M. Teixeira and Christine J. Cardin The University of Reading Chemistry Department Introduction Topoisomerases are responsible for the interconversion of the topological states of DNA generating transient single or double strand breaks in the DNA phosphodiester backbone to allow passage of one (topo I) or two (topo II) DNA strands during replication, resulting in an associated relaxation of their supercoiled state. For this reason the topoisomerases have long been an attractive target in the fight against cancer. A series of third generation anti-tumour agents have been developed in New Zealand to target malignant melanomas amongst other forms of tumour, with some inducing DNA cleavage in the presence of both topoisomerase I and II making them part of only a small range of drugs to exhibit such properties and a major step forward in the fight against cancer. Experimental We have previously show that the related 9 amino - acridine carboxamides, which are known to inhibit only topoisomerase II at the point of DNA cleaving through catalytic inhibition, can intercalate into duplex DNA with the carboxamide side chain located in the major groove binding to the N 7/O 6 of the adjacent guanine. Here we present the first examples of a range of cytotoxic agents bound at DNA junctionlike quadruplex. Crystals of the duplex d[CG(5 Br. U)ACG]2 with 9 -bromo-phenazine-1 -carboxamide in the orthorhombic space group C 222 were grown by vapour diffusion over a period of ~12 months. MAD data collection was carried out at the DESY synchrotron facility, where several wavelengths were measured to maximise the anomalous and dispersive signals. Data processing was carried out with mosflm and SCALA. Two heavy atom positions were determined by SOLVE and phase calculations carried out with MLPHARE and DM. The initial model was built ab initio into the 2Å MAD map with the map fitting program XFIT from the XTALVIEW program suite. Crystallographic refinement was carried out using SHELX 97 with SHELXPRO used for map production. Results The structure of the duplex d[CGTACG]2 bound to a range of topo poisons exhibits an unusual intercalation site and helical fraying (Figure 1) stabilised through disordered modes of drug binding and an array of cations. The intercalation cavity is formed by the strand exchange of a cytosine base rotated to pair with a guanine of a symmetry related helix at ~90° and a distance of ~15Å generating a pseudo-Holliday type junction (Figure 2). Of the cytotoxic agents shown to bind at this junction (Figure 4) the acridine based drugs have thus far only stabilised this strained system with the aid of a Mg 2+ ion, bound at the centre of the cross-over, to the four cytosine phosphates, a feature which is thus far absent for the phenazines. Two such cavities are linked through a quadruplex formed by the minor groove interactions of the N 2/N 3 guanine cavity sites (Figure 3) at an angle of ~40°, effectively Figure 2. Two views of the four way helical junction and drug cavity formed by the DNA crossover and quadruplex. For clarity the bound drugs have been removed and cobalt positions are shown in purple. (1) 9 Br-phenazine (X=N, Y=Br, Z=2) (2) DACA (X=CH, Y=H, Z=2) (3) 9 -amino. DACA (X=NH 2, Y=H, Z=2) (4) DACA 3 (X=CH, Y=H, Z=3) (5) Bis-DACA (X=9 -aminooctyl. DACA, Y=H, Z=2) Figure 4 Figure 1 Schematic view of the numbering and labelling scheme for the X-stacked junction and bound cobalt ions. Figure 3. Two illustrations of the DNA quadruplex forming the large intercalation cavity. (a) A stereoview of the cavity with drugs removed for clarity. (b) A 2Å sigma-A map showing the floor of the cavity and the minor groove interactions of the guanine N 2/N 3 positions. doubling the size of the intercalation site and allowing for a large degree of disordered binding of the drug chromophore despite the carboxamide side chain anchoring to the N 7/O 6 guanine positions in the major groove. The other end of the duplex exhibits a terminal base fraying in the presence of Co 2+ ions linking symmetry related guanine bases (see MAD pictures) intertwined through the minor groove, yielding a quasi-continuous stack. A second hydrated cobalt ion is bound to the preultimate guanine G 8 and linked through water sites to its symmetry related partner at ~7. 5Å. The structural formulae of the tricyclic drug systems which have currently been shown to help stabilise this unusual X-stacked DNA junction. Conclusions DNA junctions have been known for some time to provide high-affinity binding sites for intercalators and as this work suggests may help stabilise the X-stacked forms through charge neutralisation from their cationic side chains, as does Mg 2+. Resolution of these junctions can occur by resolvase enzymes but also some eukaryotic topo I enzymes. Human topo II has also been shown to preferentially bind to such sites. The stabilising effects exhibited by this class of compound therefore illustrates their potential to bind with DNA Three views of the 2Å MAD map used to build the initial model. Green = 1 and yellow = 2. junctions as well as duplex DNA and even strand-nicked DNA (‘hemi-intercalated’) as in the cleavable complex, suggesting a structural basis for the dual poisoning of topoisomerase I and II by this family of drugs. It must be noted however that we only obtained crystals in the presence of Co 2+ ions, and although they do not directly influence the helix cross-over and intercalation site, they are essential for this junction formation. Further Work This work suggests a possible explanation for the dual poisoning properties of certain antitumour agents, and as an extension of these studies, work looking at the formation of the cleavable complex between this family of cytotoxins and the topoisomerases has begun. Reference: Biochem. , 49, 15055 -15061, 2000 Acknowledgements: W. A. Denny (University of Auckland, NZ); P. Charlton (Xenova plc); DESY synchrotron and staff; A. K. Todd (Institute of Cancer Research, London); A. Adams (Trinity College, Dublin).
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