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Differential DNase I sensitivity of the two complementary nucleosomal DNA strands in cycloheximide-treated Ehrlich
C C Chipev1, V S Ivanova, R Tsanev
1Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
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.
Insights
Newly replicated DNA strands in Ehrlich ascites tumor (EAT) cells show asymmetric accessibility. Parental DNA fragments are more accessible near nucleosomes, suggesting chromatin interactions influence strand exposure.
Area of Science:
- Molecular Biology
- Chromatin Structure
- DNA Replication
Background:
- Understanding DNA strand accessibility is crucial for DNA replication and repair.
- Chromatin structure, including nucleosome organization, influences DNA accessibility.
- Ehrlich ascites tumor (EAT) cells provide a model for studying rapidly dividing cells.
Purpose of the Study:
- To investigate the accessibility of complementary DNA strands in newly replicated chromatin.
- To determine if chromatin structure affects the accessibility of parental DNA strands during replication.
Main Methods:
- EAT cells were cultured with cycloheximide to inhibit protein synthesis.
- DNA was labeled with 14C-thymidine (bulk) and bromodeoxyuridine/3H-thymidine (new strands).
- DNase I digestion of isolated nuclei followed by CsCl density gradient centrifugation was used to isolate newly replicated DNA.
Main Results:
- A dense fraction containing 15-20% newly replicated DNA was obtained.
- 14C-labeled parental DNA fragments complementary to 3H-nascent strands were predominantly found in the 30-50 nucleotide range.
- Simulation experiments suggest enhanced accessibility of parental strands at the nucleosomal 5'-end region.
Conclusions:
- Newly replicated DNA strands exhibit asymmetric accessibility within chromatin.
- The H2a-H2b dimer interaction site at the nucleosomal 5'-end of parental DNA may be more accessible.
- Chromatin-induced interactions likely contribute to this observed strand accessibility asymmetry.