NUCLEOSOME STRUCTURE MODULATES BENZO[A]PYRENEDIOL EPOXIDE ADDUCT FORMATION

We have studied the binding of a chemical carcinogen to DNA reconstitu ted with histone octamers to determine the effect that nucleosome stru cture has on covalent adduct formation. Reconstitution of a plasmid co ntaining the somatic 5S rRNA gene from Xenopus borealis resulted in ch aracteristic nucleosome structure, as determined by micrococcal nuclea se digestion, shifted migration in agarose gels, and hydroxyl radical footprinting. Formation of covalent adducts by benzo[alpha]pyrenediol epoxide (BPDE) occurred initially at a slower rate in reconstituted DN A than in naked plasmid, but after 2 h the total adduction levels (add ucts/plasmid) were equal in both samples. Analysis of adduction at the sequence level by primer extension indicated that, after a 2-h BPDE r eaction, the degree of adduction within the 5S rRNA nucleosome was sup pressed by approximately 50% compared to naked DNA. The rotational set ting of the guanines on the helix did not explain the level of adducti on observed, since guanines in close proximity to the histone core wer e equally susceptible to adduction as guanines on the outer nucleosome surface. At early reaction times with BPDE, however, sequences near t he 5S nucleosome dyad, where known modulations in the minor groove wid th occur, were the least susceptible to adduction. These results indic ate that the structural features of DNA assembled into nucleosomes con tribute to the susceptibility of the DNA to modification by BPDE.