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Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells
Authors:C C Chipev  V S Ivanova  R Tsanev
Institution:Institute of Molecular Biology Bulgarian Academy of Sciences , 1113 , Sofia , Bulgaria
Abstract:Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted 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 bromodeoxyu ridine 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 replica ted 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 tobe induced by interactions in the chromatin.
Keywords:
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