DNA studies within the School of Chemistry

1. The binding modes of intercalating experimental anti-tumour agents to DNA has proved far more complex than the simple model of intercalation represented in most texts. Works carried out here have revealed the disordered nature of intercalating binding for a bis-intercalator, where we have shown it to specifically target a larger intercalation site formed at the junction of four helices. The drug is therefore observed to thread through the enlarged binding pocket, rather than the more commonly The role of metal ions in determining the solution conformation of the Holliday junction is well established, but to date the picture of metal ion binding from structural studies of the four-way DNA Holliday junction is very incomplete. Here minor groove. The Sr2+ ions occupy the TC/AG, GG/CC and TA/TA sites in the minor groove, giving ten positions forming two spines of ions spiralling through each arm of the X-stacked structure. Both structures show an opening of the minor groove face Accumulating evidence within the literature to date regarding the biological significance of quadruplex DNA, provides compelling support for its highly stable nature of this quadruplex hairpin. Studies into the addition of more toxic metal species to quadruplex DNA have revealed results thus far for nickel, where it is observed to distort the hairpin existence and functions within biological systems, making it an enticing therapeutic target. The synthetic DNA heptamer d(GCATGCT) under structure through the covalent binding of the two available guanine N7 positions and with four bound solvent oxygens, the ion attains a complete octahedral coordination agents and the multiple modes of binding observed both for the chromophore and more importantly the anchoring sidechain. Atomic and high resolution studies have revealed the messy multiplicity now known to affect the properties of accepted sandwich style binary modes of binding. The identification of sequence specific binding sites for experimental anti-tumour agents is now undertaken prior to crystallisation studies through the technique of competitive dialysis. The we present two refined structures of the Holliday junction formed by d(TCGGTACCGA) in the presence of Na+ and Ca2+ ions and separately Sr2+ ions, to resolutions of 1.85Å and 1.65Å. This sequence includes the ACC core found to of the junction of 8.4° in the Ca2+ and Na+ containing structure and 13.4° in the Sr2+ containing structure. The crossover angles at the junction are 39.3° and 43.3° respectively, and a relative shift in the base pair stack alignment of certain ion stabilisation conditions, forms a hair-pin looped quadruplex, composed of four G/C base pairs, as opposed to the more orthodox G tetrads. Thus far the stabilisation of shell. A C/G quadruplex is still maintained through the binding of a central sodium ion which tethers the bases in a similar fashion to that observed for many G tetrads. The aim of this study is therefore to catalogue the interactions of a series these chemotherapeutic agents. Further to the model of intercalation into duplex DNA, our studies have focussed on the modes of binding to higher order DNA structure as a potential target for many highly active clinical agents. These studies have yielded a previously unobserved mode of methodology involves the soaking of several selected DNA sequences in a low concentration of drug solution, resulting in the drug binding to those preferred sequence sites, and is studied through UV spectrometry. Ref: JMB, (2002), 323, p167-171. Many thanks go to Dr. Peter Charlton of this unusual motif has been observed in the presence of magnesium, cobalt hexamine and barium where each hydrated ion is observed to bind within solvent cavities to phosphate oxygens. X-ray measurements of this structure have been carried out to atomic resolution, revealing the promote spontaneous junction formation. The Na+ sites, the most convincing observations in junctions to date, are on either face of the junction crossover region, and stabilise the highly ordered hydration within the arms. The four Ca2+ sites in the same structure are at the CG/CG steps in the the arms of 2.3Å is observed for the strontium structure. Overall these results provide an insight into the so-far elusive stabilising ion structure of the DNA Holliday junction. Ref: JMB, (2003), 327, p97-109 Many thanks go to the AICR for support in this project of metals with DNA and hence relate these crystallographic studies to the toxic nature of the cations. Ref: Nucleic Acids Research, (2003), 31 (3), p844-849. Strontium is known to mimic calcium within natural systems, making it a good candidate for derivatising calcium binding macromolecules. It is ideally suited for use with synchrotron radiation sources as an anomalous scatterer, having biological function of its own, it follows the metabolic pathways of calcium and behaves similarly, although it is not homeostatically controlled. Structurally, the strontium ion is larger than the calcium ion, thereby requiring a slightly wavelengths achievable at most modern facilities utilising the Sr K-edge at ~0.77Å. We therefore present the crystal structure of the four-way Holliday junction formed by the DNA sequence d(TCGGTACCGA) solved through the multi-wavelength anomalous dispersive (MAD) phasing of the hydrated group II larger binding site within a macromolecule. However their hydration numbers are the same, with a value of 29, illustrating further similarities between the chemistries of both cations and a feature that provides each with a similar charge stabilising potential. In conclusion we propose strontium as a good metal ion, strontium, to a resolution of 1.6Å, and final refinement to 1.5Å. Within the PDB to date there are numerous examples of macromolecular calcium binding derivatives, whereas there are surprisingly few examples illustrating strontium binding. Although strontium has no direct candidate for derivatising calcium binding macromolecules for use in anomalous diffraction measurements. The figure illustrates the MAD phases achieved from strontium using the tuneable facilities at DESY Hamburg (beam line BW7A). Ref: To be published. DNA studies within the School of Chemistry J.H. Thorpe, S.C.M. Teixeira, B.C. Gale, Y. Gan, M.I.A.A. Moraes and C.J. Cardin The DNA Holliday Junction Quadruplex DNA Drug Binding Studies – Crystallography and Competitive Dialysis X-ray Anomalous Diffraction Studies