Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

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.

From Chemical Embryology to Nucleosome Patterning - an interview with Roumen G. Tsanev

“…We also found bilateral dispersive nucleosome segregation in groups of several adjacent nucleosomes distributed between the two daughter DNA molecules (Pospelov et al, 1982) The distribution of old and new histones between the two DNA strands could be explained by differences in the interaction of histones with the two complementary DNA strands. We were then able to demonstrate that the nucleosomal histone core, when in the chromatin, is slightly asymmetrically placed in the region where the H2A-H2B pairs have been shown to interact with DNA (Chipev et al, 1987). Such an asymmetry was confirmed by later publications and by the fact that an alternating asymmetrical protection of DNA in nucleosomes was seen inside the chromatin higher-order structure (Staynov, 2000).…”