Differential formation of O6-ethylguanine in the DNA of rat brain chromatin fibers of different folding levels exposed to N-ethyl-N-nitrosourea in vitro.

Extended (histone H1-depleted), 11-nm-thick chromatin fibers and condensed 25- to 35-nm-thick chromatin fibers, representing the first and second level of DNA folding in chromatin, respectively, as well as nucleosome core particles, were isolated from fetal rat brain cells and briefly exposed to N-ethyl-N-nitrosourea (EtNU) in vitro. The O6-ethyl-2'-deoxyguanosine (O6-EtdGuo):2'-deoxyguanosine (dGuo) molar ration in DNA enzymatically hydrolyzed to 2'-deoxynucleosides was determined by competitive radioimmunoassay using an anti-O6-EtdGuo monoclonal antibody with an affinity constant for O6-EtdGuo of approximately 2 X 10(10) liters/mol. In comparison to naked DNA (O6-EtdGuo:dGuo relative value, 1.0) the O6 atom of guanine proved to be increasingly protected from ethylation by EtNU in the DNA of the histone H1-depleted chromatin fibers (relative value, approximately 0.6) and of the condensed chromatin fibers (relative value, approximately 0.4). From the O6-EtdGuo:dGuo relative values obtained for the DNA of nucleosome core particles (approximately 0.5) and of H1-depleted chromatin fibers (approximately 0.6), it follows that the accessibility of the O6 atom of guanine to the electrophilic ethyldiazonium ion generated from EtNU in internucleosomal DNA (protein free) is about twice that found in nucleosomal DNA. The overall O6-EtdGuo:dGuo molar ratio in the DNA of H5 rat hepatoma cells exposed to EtNU in vitro was similar to that of the DNA of the condensed 25- to 35-nm chromatin fibers. Since the bulk of genomic DNA is organized in the form of these chromatin fibers, the overall degree of intercellular O6-EtdGuo formation appears to be mainly determined by this folding level of DNA, and not, or only to a low degree, by the DNA folding levels of higher-order chromatin structures such as those found in large loops or domains of chromatin fibers within the cell nucleus.