Graduate Thesis Or Dissertation

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

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