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DNA replication intermediates in whole HeLa cells and isolated nuclei
Biochimica Et Biophysica Acta
|April 19, 1977
Summary
Newly synthesized DNA fragments, termed primary DNA pieces, were investigated in HeLa cells. These fragments are rapidly incorporated into larger DNA molecules, indicating their role in DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Understanding DNA replication is crucial for cell division and genetic stability.
- Replicative intermediates are transient DNA structures formed during DNA synthesis.
Purpose of the Study:
- To investigate the size and fate of nascent DNA fragments (primary DNA pieces) during replication.
- To compare DNA replication intermediates in intact cells versus isolated nuclei.
Main Methods:
- Pulse-labeling of HeLa cells and isolated nuclei with [3H]thymidine.
- Analysis of DNA fragment size using sedimentation velocity (S values).
- Chase experiments to track the incorporation of labeled DNA into larger molecules.
Main Results:
- In intact cells, primary DNA pieces were 90-160 nucleotides long and rapidly incorporated into high molecular weight DNA (30-70 S).
- In isolated nuclei, primary DNA pieces were ~200 nucleotides and also rapidly incorporated.
- In vitro, some primary DNA pieces elongated to 2000-3000 nucleotides before joining larger DNA structures.
Conclusions:
- Primary DNA pieces are short, newly synthesized fragments that are quickly processed and integrated into the replicating genome.
- Cellular environment influences the size and processing of these early replication intermediates.