BIOPHYSICAL STUDIES OF OLIGOPURINE TRACTS

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Oligopurine sequences have been shown to be versatile elements in vitro, capable of assuming several different alternative DNA conformations. A recent search of over one half million nucleotides of published DNA sequences has shown that long tracts greater than 10 base pairs of contiguous purine residues are greatly and consistently overrepresented in eukaryotic, but not prokaryotic, organisms. The hypothesis motivating the work presented in this proposal is that such long oligopurine tracts have physical characteristics that are different from bulk DNA, and that the eukaryotic cell can take advantage of these characteristics to exert biological effects. The purpose of the proposed research is to understand the physical and, to an extent, the biological properties of oligopurine- oligopyrimidine [(Pu-Py)n] regions in polymeric DNA. The proposal begins with a survey of the circular dichroism spectra of a number of defined-sequence polydeoxynucleotides under a variety of solution conditions in order to grasp where the beginning is, -i.e., at what point are (Pu-Py)n regions in a recognizably different conformation than bulk DNA. The line of investigation then continues with the quantitative determination of a property of the sequences that is likely to be important to the role they play in vivo: their ability to affect nucleosome formation. This will be done using a competitive nucleosome reconstitution assay, developed by our laboratory, that allows the determination of the difference in the free energy of nucleosome formation between competing polynucleotides. Collaborative transient electric birefringence studies, measuring the bending stiffness of the synthetic polymers, will then be performed and correlated to the structural changes seen in CD studies and the functional changes seen in nucleosome reconstitution assays.