In vitro binding of N-acetoxy-N-2-acetylaminofluorene to DNA in chromatin

Abstract

The binding of N-acetoxy-N-2-acetylaminofluorene to DNA in native and partially dehistonized chicken erythrocyte chromatin was studied. The amounts of carcinogen bound to DNA were measured, after removal of proteins with phenol, by using the absorption ratio A305/A260 or by counting the radioactivity of 14C-labeled carcinogen. Measurements of uncovered zones of DNA in chromatin were made by comparison of results obtained with free DNA and with chromatin at increasing ratios of carcinogen/nucleotide. The proportion of DNA accessible to the carcinogen was found to be 15% in native chicken erythrocyte chromatin and about 22% in native calf thymus chromatin. The amount of accessible DNA increases to 55% in chicken erythrocyte chromatin depleted of histones H1 and H5. Formaldehyde unwinding performed on DNA extracted from chromatin after modification showed an increasing number of defects in the double helix with the amount of DNA-fixed carcinogen. At high ratios of carcinogen/nucleotide, the recoveries of DNA (by phenol method) and of histones (by acidic extraction) decreased with increasing ratios. This suggests a covalent linkage between proteins and DNA.