This is a competing renewal application which characterizes the range of structure and flexibility associated with DNA polymorphism in aqueous solution and attempts to define the fundamental principles associated with the binding of antitumor antibiotics antitumor proteins to their sequence dependent target sites on DNA. Structural feature are determined following application of two dimensional homonuclear and heteronuclear N experiments combined with structure reconstruction algorithms while base pair opening kinetics are monitored by hydrogen exchange measurements on the resolved imino protons of the DNA oligomers and their complexes. The ongoing research program on helical errors in DNA is expanded to address the pairing alignment at purine-purine mismatch sites and the stacked-looped out equilibrium at multiple deletion bulge sites in mixed sequence DNA, as well as in (A)n . (T)n tracts and the dinucleotide repeat of Z-DNA. The details of chain reversal in hairpin loops will be characterized further by insertion of cytotoxic agents 5-fluorouracil and arabinosyl cytosine within the loop and monitoring the consequences of base ionization and hydrogen bonding potential on loop conformation and dynamics. The ongoing studies on 06-alkylguanosine modified DNA oligomers will be extended to the characterization of this carcinogenic lesion when inserted in the 12th codon of H-ras where it is responsible for oncogenic transformation. A group of new projects addressing DNA polymorphism have been initiated and range from studies on parallel-stranded duplex telomere sequences, triple helices and DNA pseudoknots in aqueous solution. Our goal is to characterize the base pairing alignments and helical parameters that define these higher order DNA structures. Parallel measurements are being undertaken on B-Z junctions and three-stranded junctions to address questions related to base pairing, helix orientation and flexibility at the junction site and the role of metal ions in modulating the structure and stability of the junction. We are currently characterizing the complexes of the antitumor antibiotics luzopeptin (a bisintercalator) and CC-1065 (a covalent binder) with their sequence specific DNA binding sites in order to identify intermolecular contacts involved in drug-DNA recognition. The solution structures of these complexes will be defined by combined NMR- structure reconstruction techniques prior to investigation of conformational changes upto a helical turn away from the binding site established from footprinting experiments. Finally, we propose to characterize the solution structures of the holo- and apo- forms of the antitumor protein neocarzinostatin. The role of neocarzinostatin cofactor in ligand-protein and ligand-DNA interactions will be pursued in the present application.
NIH ExPorter
NIH ExPorter