An experimental investigation of the interactions of stable, branched DNA oligonucleotide complexes (DNA junctions) with various ligands is proposed. The rationale for this study stems from data suggesting that sites of branching in DNA may be capable of very tight binding of ethidium ion as well as other intercalaters. Branched forms of DNA, such as occur naturally as intermediates in recombination for example, might represent major targets for agents that alter normal regulation or induce mutations in cells. How ligands alter the structure and dynamics of branched molecules is of interest in its own right. First, a series of drug molecules will be screened for their potential to bind selectively at the site of branching in three and four arm junctions. Gel foot-printing analysis will be used to establish the degree of this selectivity, the symmetry properties and binding constant(s) of the interaction. Protein ligands and enzymes will also be investigated, using electrophoretic methods. Binding proteins will be selected by means of Alberts columns prepared with junctions. Antibodies to DNA junctions will also be sought in an effort to find reagents selective for the presence of recombination intermediates in cells. Second, the structure of selective drug-junction complexes will be studied, using IH and 31P NMR. Psoralen photoreaction will be used to investigate unliganded and liganded junctions. These studies should elucidate the structure and structural dynamics of junctions, as wll as the nature of the interaction of drug and protein molecules with them. The properties of junctions as substrates for DNA enzymes will also be studied, with and without ligands.
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