NUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE COPPER ION

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DESCRIPTION: (adapted from applicant's abstract) The goals of the proposed research are 1) to design ribooligonucleotide analogs which inhibit transcription by hybridizing to initiation sites of RNA synthesis; 2) to use regulatory protein-1,10-phenanthroline chimeras to identify undiscovered DNA binding sequences in genomic DNAs. Both research directions stem from Dr. Sigman's continuing studies on the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu). The transiently unwound DNA formed at transcription start sites can be hybridized by short ribooligonucleotide analogs complementary to the template strands of promoters. The studies with the UV5 and trpEDCBA promoters will be extended by examining the stability of the open complexes as a function of nucleoside triphosphate concentrations, chemically modified backbones, and sugar moieties. The aims of these studies will be a) to identify the accessible sequences within the template strand of this transient intermediate, and b) to increase the affinity and promoter specificity of the oligonucleotide inhibitors. The homology between the open complexes of eukaryotes and prokaryotes will be investigated with special attention to the role of ATP in making single-stranded DNAs accessible in eukaryotic systems. The RNA polymerase assisted formation of unwound DNA makes the use of short oligonucleotides possible in the design of gene specific inhibitors of potential pharmacological significance. The site-specific scission of regulatory protein-OP chimeras permits the analysis of nucleic acid binding by proteins in vitro. Targeted scission also provides an approach for identifying the binding sites of regulatory sequences in genomic DNA. An analytical method for detecting these sites of scission has been devised which would allow the identification of sites in the E. coli and yeast genomes. In addition, new methods of preparing OP-chimeras based on the chemistry of scission of proteins by OP-Cu have been devised. These methods of finding regulatory motifs will be tested with the E. coli trp repressor and cAMP binding proteins and the zinc cluster proteins of GAL4, PUT3 and PPR1 of yeast. These methods could help characterize genomes in non-coding regions.