Effect of N-2-acetylaminofluorene modification on the structure and template activity of DNA and reconstituted chromatin

Abstract

This study compares the effects of in vitro modification of native duck reticulocyte DNA by [14C]-N-acetoxy-2-acetylaminofluorene in terms of alterations in DNA secondary structure, ability to reconstitute nucleosome structures in chromatin, and template activity for in vitro transcription. In contrast to the control native DNA, the carcinogen-modified DNA was susceptible to partial digestion by the single-strand-specific endonuclease S1. Depending on the particular conditions, for every [14C]-N-2-acetylaminofluorene residue released, about 5 to 35 base pairs of DNA were also released during the S1 nuclease digestion. Chromatin was reconstituted in vitro utilizing [14C]-N-2-acetylaminofluorene-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 gradient density centrifugation as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. The carcinogen-modified DNA and also chromatin reconstituted from this DNA showed, however, marked reductions in their abilities to serve as templates for transcription with Escherichia coli RNA polymerase. These results suggest that the covalent binding of N-2-acetylaminofluorene to DNA produces localized regions of denaturation in the DNA and that this is associated with a marked impairment in template activity during transcription. This modification, however, does not grossly affect the ability of the DNA to interact with chromosomal proteins to form apparently normal nucleosome structures.