The application of psoralens to the study of DNA structure, function and dynamics

Abstract

A series of six nitroxide spin-labeled psoralens were designed, synthesized and tested as probes for DNA dynamics. The synthesis of these spin-labeled psoralen derivatives and their photoreactivity with double-stranded DNA fragments is described. The spin labels (nitroxides) were demonstrated to survive the uv irradiation required to bind the probe to the target DNA. EPR spectra of the photobound spin-labels indicate that they do not wobble with respect to the DNA on the time-scales investigated. The author has used psoralen modified DNA as a model for the study of DNA repair enzyme systems in human cell free extracts. He has shown that damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is "repair synthesis" and not an aberrant DNA synthesis reaction potentiated by deformation of the DNA by adducts. He has found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach he has obtained evidence that this in vitro system is capable of removing psoralen cross-links as well. Reported here are synthetic methods that make use of high intensity lasers coupled with HPLC purification to make homogeneous and very pure micromole quantities of furan-side monoadducted, cross-linked, and pyrone-side monoadducted DNA oligonucleotide. These molecules are currently being studied by NMR and X-ray crystallography. The application of the site-specifically psoralen modified oligonucleotide synthesized by these methods to the construction of substrates for the investigation of DNA repair is also discussed.