D(GPG) IN CRUCIFORM LOOPS AS TARGETS FOR CISPLATIN

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THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Here, we propose that distorted DNA structures, specifically the loops of cruciforms, may render certain d(GpG) dinucleotides more susceptible as targets for modification by the antitumor drug cis- diamminedichloroplatinum (II) (cis-DDP, or cisplatin). This is a radical departure from the standard paradigm that cis-DDP binds to d(GpG) dinucleotides in linear DNA and subsequently distorts the DNA duplex. Our model was derived from preliminary studies which show that cis-DDP, but not its clinically inactive trans-isomer, can shift the dynamic equilibrium between duplex DNA and a cruciform, and a recent NMR structure of a platinated DNA hairpin. The role of these structural perturbations may he to provide greater specificity to the drug by setting up the stereochemical environment that is more amenable to the structure of the cross-linked adduct. We estimate that the number of d(GpG) sites in a human genome that lie in cruciform forming sequences (about 5,000) is similar to the number of drug molecules found in a cell during treatment (about 1,000). The proteins that recognize platinum adducts in DNA (particularly the high mobility group or HMG classes of proteins) may actually be recognizing a cis-DDP stabilized cruciforms. These proteins have been shown to bind to synthetic four-way junctions, which form at the base of cruciforms. In this application, we propose studies to provide further support of this new model. We propose to: i) determine whether d(GpG) sites in cruciform loops are significantly more susceptible to cis-DDP binding compared to the same sites in linear duplex DNA by comparing the rate of platinum reaction at a d(GpG) site in linear versus extruded cruciform DNAs; ii) determine the degree to which cis-DDP helps to stabilize the extruded cruciform by two-dimensional gel analysis of topoisomers of cruciform containing plasmids; and iii) determine whether HMG-I--a protein which is elevated in certain cancerous cells--recognizes and binds to cis-DDP stabilized cruciforms by comparing the binding of this protein to d(GpG) sites in cruciform and linear DNAs.