The overall purpose of this project is to help elucidate the relationship between structure and function of viral and ultimately cellular nucleoprotein complexes. In addition to biochemical approaches, I plan to take advantage of the permeability of cells and virus particles to furocoumarin derivatives such as 4,5',8-trimethylpsoralen. Psoralens are currently used in the treatment of dermatological disorders such as vitiligo and severe forms of psoriasis, although their primary mode of action has not yet been established. These componds intercalate readily into the double-stranded helix of DNA, and in the presence of long wavelentth ultraviolet light form covalent photoadducts (monoadducts) via a cyclobutane ring with thymine residues. If two thymines are located in adjacent base pairs on opposite strands, a second photoevent can result in cross-link formation between the two strands. Because of both the specificity of psoralen compounds for accessible regions of DNA such as internucleosomal bridges, and the ability to study intracellular and viral structures in vivo without prior disruption of the biological system this approach will allow a detailed evaluation of cellular and viral nucleosome arrangements and of virion (virus particle) nucleoprotein structure. The projects described in this application can be divided according to the approach and system employed: (1) The structure of intracellular SV40 nucleoprotein complexes will be analyzed in order to study the relationship between nucleoprotein structure of the origin regions, the function of tumor antigen, and the regulation of transcription and replication, employing both in vivo cross-linking and in vitro tumor antigen binding systems. (2) The structure and packaging of the DNA within SV40 mature virion and previrion particles and within lambda bacteriophage particles will be investigated by both saturation kinetics and by specific activity determinations of restriction fragments following the """"""""in vivo"""""""" photoaddition of radioactive psoralen derivatives. (3) A method for fine structure mapping of psoralen photoadducts will be developed which should enable the adduct positions to be read from a DNA sequencing electrophoretic gel of exonuclease-resistant 32P-end-labeled restriction fragments.
| Kondoleon, S K; Kurkinen, N A; Hallick, L M (1989) The SV40 nucleosome-free region is detected throughout the virus life cycle. Virology 173:129-35 |
| Ostrander, E A; Kurkinen, N A; Hallick, L M (1988) DNA structural alterations in the SV40 enhancer region are retained in vivo. Virology 165:274-7 |
| Ostrander, E A; Robinson, G W; Isaacs, S T et al. (1986) The site-specific inhibition of Bgl I cleavage by psoralen photoadducts. Photochem Photobiol 44:21-9 |
