The life cycle of HIV, the etiological agent of AIDS, is directed by proteins that control its integration into the host DNA to form HIV chromatin, and all subsequent steps. Means of inhibiting HIV integration, replication, and of inactivating HIV genomes already integrated into the host must be sought. This will require an understanding of the structure and function of the DNA-protein complexes that control these processes. A major effort of this laboratory is to understand the 3-dimensional architecture of DNA- protein complexes. Recently it has been shown that the sequence of bases along a DNA may cause it to have a complex curvature in 3-dimensions. Thus to understand how proteins bind to HIV DNA it must first be determined whether each segment of HIV DNA is straight or curved-- in the absence of bound proteins. Here this information will be provided through a quantitative curvature map of HIV DNA. Powerful electron microscopic (EM) and computer methods have been developed for measuring DNA curvature. Restriction fragments are prepared and the fragments labelled at one specific end with avidin to provide an absolute orientation. From micrographs of hundreds of molecules a computer-generated map is produced that describes the curvature at each point on the DNA by a mathematical parameter. From the micrographs the shape of highly curved segments can be described. This map will be correlated with data from gel electrophoresis. The EM data will also reveal sites along the DNA that are unusually stiff and thus might exclude the binding of proteins such as the histones that wrap DNA around themselves. The curvature map will provide an important new physical map of HIV DNA. With this information, the positioning of nucleosomes in reconstituted HIV chromatin will be mapped and related to regions of high or low DNA curvature. Binding sites for topoisomerase II and sites on the DNA that are cleaved in the presence of drugs will be determined since this may represent a future portal for inactivation of integrated HIV genomes. Finally specific transcription factors, in particular Spl will be examined bound to HIV DNA and chromatin.
