The photoaddition of trimethylpsoralen to Drosophila melanogaster nuclei: a probe for chromatin substructure

Abstract

Derivatives of the furocoumarin, psoralen, can penetrate intact cells or nuclei and cross-link opposite strands of the chromosomal DNA under the influence of long wave-length UV light. The potential of trioxsalen (4,5'',8-trimethylpsoralen) as a probe for chromatin structure was investigated. The DNA in both embryo nuclei and tissue culture cells from D. melanogaster was .apprx. 90% protected from trioxsalen binding relative to purified DNA. Digestion of trioxsalen-treated nuclei by micrococcal nuclease and gel electrophoresis of the resulting DNA gave the same type of band pattern characteristic of native, untreated nuclei after digestion. Nuclease digestion was therefore used to examine the distribution of bound trioxsalen in the DNA. The resulting DNA fragments were analyzed by radioactivity measurements and quantitative EM. The nuclease cleaved intact photoreacted nuclei in such a way that preferential excision of trioxsalen containing regions of the DNA occurred, but when acting upon purified DNA that contained bound trioxsalen, it attacked the trioxsalen-free regions preferentially. Trioxsalen binds at the sites corresponding to the regular nuclease sensitive regions of the chromatin in nuclei.