DNA STRUCTURE AND GENE REGULATION

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The mechanism of genetic regulation at the molecular level will be studied in vitro and in vivo with special emphasis on the role of DNA properties and conformations as determinants of specificity. Unusual DNA conformations (left-handed Z-DNA, cruciforms, structures adopted by oligopurine.oligopyrimidine sequences) will be investigated with synthetic oligonucleotides, restriction fragments, and recombinant plasmids. Conformational features will be analyzed with a variety of probes including nucleases, chemical modifications, supercoil relaxation determinations, crosslinking studies, electron microscopy, and immunological approaches. The structure and properties of unusual DNA conformations will be studied in vitro with oligonucleotides per se and with recombinant plasmids. A range of structural, thermodynamic, kinetic, molecular modeling, and hydrodynamic properties will be evaluated. Model studies will be undertaken on a consortium basis for evaluating the characteristics of these probes at the base pair level. The interactions of specific proteins with unusual DNA structures will be investigated in vitro and in vivo. The binding properties of the E. coli recA protein and the T4 gene 32 protein in vivo and in vitro with Z-DNA will be established using several different assays. The influence of neighboring left-handed Z-DNA tracts on the interaction of the E. coli lac repressor with its operator and the influence of the neighboring promoter will be studied as a function of negative supercoiling. The types of unusual conformations adopted by genetic control regions of SV40 and JC virus in the presence and absence of specific proteins will be studied. The in vivo existence and function of unusual DNA conformations, including Z-DNA, will be evaluated with new systems developed for this purpose. A plasmid incompatibility assay appears particularly promising in preliminary investigations. The HhaI and EcoRI methylases will be established and utilized in vivo as conformational probes. The role of Z-DNA in homologous recombination and in in vivo gene expression assays will be undertaken. An understanding of the details of genetic expression is fundamental to our ultimate comprehension of normal cellular growth and division as well as disease syndromes characterized by unregulated growth such as cancer.