Site-specific aflatoxin B₁ adduction of sequence-positioned nucleosome core particles

Graduation date: 1989The question of how the presence of nucleosomal\ud packing of DNA modifies carcinogen interaction at\ud specific sites cannot be answered by studies on whole\ud chromatin or bulk nucleosomes because of the\ud heterogeneity of DNA sequences in the particles. This\ud problem was circumvented by constructing nucleosomes that\ud are homogenous in DNA-histone contact points. A cloned\ud DNA fragment, containing a sea urchin 5S gene which\ud precisely positions a histone octamer was employed. By\ud using 32P end-labeled DNA and genotoxins that allow\ud cleavage at sites of attack, the frequency of adduction\ud at every susceptible nucleotide can be determined on\ud sequencing gels. The small methylating agent dimethyl\ud sulfate (DMS) and the bulky alkylating agent aflatoxin\ud B₁- dichloride (AFB₁-C1₂) were used to probe the influence\ud of DNA-histone interactions on DNA alkylation patterns in\ud sequence-positioned core particles. I find DMS to bind\ud equally to naked or nucleosomal DNA, in a quantitative\ud 1:1 ratio. In contrast, AFB₁-C1₂ binding is globally\ud suppressed 2.4-fold in nucleosomes, but the DNA is more\ud accessible to AFB1-C1₂ adduction in regions near the\ud particle boundary. I observe no other histone-imposed\ud localized changes in AFB₁ -C1₂ sequence specificity.\ud Further, sites of DNase I cleavage or proposed DNA\ud bending show neither enhanced or reduced AFB₁-C1₂\ud adduction to N7-guanine. Since AFB₁-C1₂ binding sites\ud lie in the major groove, nucleosomal DNA appears\ud accessible to AFB₁-C1₂ at all points of analysis, but\ud with an access which is uniformly restricted in the\ud central 100 nucleotides of the core particle. The data\ud available do not indicate further localized or sitespecific\ud perturbations in DNA interactions with the two\ud carcinogens studied