Differential DNase I sensitivity of the two complementary nucleosomal DNA strands in cycloheximide-treated Ehrlich ascites tumor cells

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibited protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with 14C-thymidine and the newly synthesized strands - with bromodeoxyuridine and 3H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15-20% newly replicated DNA. Analysis of the distribution of 14C-labeled parental DNA fragments complementary to the 3H-nascent strand has shown that the 14C-labeled fragments prevail in the region of 30-50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5'-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems to be induced by interactions in the chromatin.