Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: Relation to chromatin structure and reconstitution

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S₁ than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the ‘open’ regions of chromatin, and 35% to ‘closed’ regions.These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.