Nucleosome structure modulates benzo[a]pyrenediol epoxide adduct formation

We have studied the binding of a chemical carcinogen to DNA reconstituted with histone octamers to determine the effect that nucleosome structure has on covalent adduct formation. Reconstitution of a plasmid containing the somatic 5S rRNA gene from Xenopus borealis resulted in characteristic nucleosome structure, as determined by micrococcal nuclease digestion, shifted migration in agarose gels, and hydroxyl radical footprinting. Formation of covalent adducts by benzo[a]pyrenediol epoxide (BPDE) occurred initially at a slower rate in reconstituted DNA than in naked plasmid, but after 2 h the total adduction levels (adducts/plasmid) were equal in both samples. Analysis of adduction at the sequence level by primer extension indicated that, after a 2-h BPDE reaction, the degree of adduction within the 5S rRNA nucleosome was suppressed by approximately 50% compared to naked DNA. The rotational setting of the guanines on the helix did not explain the level of adduction observed, since guanines in close proximity to the histone core were equally susceptible to adduction as guanines on the outer nucleosome surface. At early reaction times with BPDE, however, sequences near the 5S nucleosome dyad, where known modulations in the minor groove width occur, were the least susceptible to adduction. These results indicate that the structural features of DNA assembled into nucleosomes contribute to the susceptibility of the DNA to modification by BPDE.