Covalent binding of the carcinogen benzo[a]pyrene diol epoxide to Xenopus laevis 5 S DNA reconstituted into nucleosomes.

Abstract

The sequence-specific interactions of bulky carcinogens with purified DNA might reasonably be expected to be altered when the DNA is organized into chromatin. We have approached this subject by studying the covalent binding of a potent carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) to nucleosomal and free DNA, using a defined fragment of DNA derived from the 5'-end of the 5 S rRNA gene of Xenopus laevis, reconstituted into nucleosomes by salt exchange with unlabeled chicken mononucleosomes. Micrococcal nuclease and hydroxyl radical "footprinting" experiments demonstrated the formation of a uniquely positioned nucleosome covering the transcriptional start point of the gene. The reconstituted nucleosomes or control DNA samples were modified with BPDE-I. DNA was repurified from the nucleosomal and control modification reactions and then irradiated with laser light at 355 nm, causing strand breaks at the positions of adducts. Nucleosomal DNA exhibited a marked decrease in the level of carcinogen binding, especially in the central 80-90 base pairs of the nucleosomal region. Interestingly, although overall binding was inhibited about 2-fold, the sequence-specific pattern of binding to deoxyguanosine residues seen with purified DNA was maintained in the nucleosomal DNA.