The major goals of the proposed research are (1) a better understanding of the relationship between base sequence and DNA structure and (2) elucidation of the means by which specific DNA-binding proteins induce alterations in DNA structure. Alteration in helix structure, as a consequence of specific protein-DNA interactions, are generally believed to play a major, albeit undefined, role in a wide variety of processes, including DNA processing, the regulation of transcription, and DNA packaging. A knowledge of the molecular mechanisms of such processes is of fundamental importance to our general understanding of gene expression. In studying the sequence-dependence of helix structure, advantage will be taken of the observation that certain sequences give rise to curvature of the helix axis. A number of models have been proposed to explain the observed curvature; however, these models have not been directly testable due to the paucity of quantitative experimental data regarding curvature. One of the primary aims of this proposal is to establish a quantitative relationship between helix curvature and base sequence. The technique of differential decay of birefringence (DDB) will be utilized thoughout the proposed investigations. The DDB measurements (sensitive to differences in DNA length as small as one-percent) will be used in conjunction with DNA ring-closure measurements and oligodeoxynucleotide synthesis to carry out essentially all of the studies comprising this proposal. Three specific protein-DNA complexes will be used as paradigms for the investigation of protein-induced structural alterations of the helix axis. The first protein, EcoRI endonuclease, is an example of a DNA processing enzyme, and its complex with DNA is the first to have been visualized by X-ray diffraction methods. The second protein, the catabolite gene activator protein, is of major importance in the regulation of transcription in E coli. The third protein, HU of E coli, is involved in DNA organization and/or packaging. For each of these three systems, protein-induced curvature has been proposed on the basis of X-ray diffraction studies, changes in linking number, and/or behavior on gels. Using DDB and ring-closure measurements, protein-induced alterations in helix structure will be evaluated for each of the systems, as will the relationship between curvature, if present, and base sequence. This study will therefore provide an important link between structures deduced from diffraction studies and those existing in solution.
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