In vivo and in vitro investigations are proposed on non-B DNA structures including intramolecular triplexes and other non-B right-handed conformations at oligopurine.oligopyrimidine tracts. Studies will be conducted on naturally occurring pur.pyr sequences to attempt to establish biological functions in prokaryotic and eukaryotic cells. Also, systems will be investigated where synthetic pur.pyr tracts are cloned specifically at certain loci to attempt to elucidate biochemical behaviors. Biological systems include replication and transcription. Direct determinations will be established to evaluate the existence and properties in vivo of non-B DNA oligopurine.oligopyrimidine structures in plasmids and chromosomes. Several probes will be utilized including UV photofootprinting, KMnO4, and methylase expression systems. In vitro emphases include: elucidation of other types (non- triplex) of non-B DNA structures at pur.pyr tracts; conditions which stabilize these structures and their mechanisms of formation; enzymes (such as methylases, restriction enzymes) as conformational probes; the effect of flanking sequences on thermodynamics, kinetics, and structures; the effect of methylation of C residues; and the isolation and characterization of proteins that specifically bind these non-B conformations. These studies should provide significant insight into the biological and chemical properties of pur.pyr tracts which are found in a variety of biologically important systems.
