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.
Chipev
C C
CC
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
Ivanova
V S
VS
Tsanev
R
R
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000648:ultrastructure; D003513:Cycloheximide / Q000494:pharmacology*; D004249:DNA Damage; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D009707:Nucleosomes / Q000187:drug effects* / Q000648:ultrastructure
D002843:Chromatin; D004273:DNA, Neoplasm; D006657:Histones; D009707:Nucleosomes; D003513:Cycloheximide; D003850:Deoxyribonuclease I