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Related Experiment Videos

A permeable cell system for studying DNA replication in synchronized HeLa cells.

S Seki, M Lemahieu, G C Mueller

    Biochimica Et Biophysica Acta
    |February 10, 1975
    PubMed
    Summary

    HeLa cells treated with hypotonic buffer can replicate DNA using external nucleotides. This DNA synthesis occurs at pre-existing active sites and is confined to the nucleus.

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    Area of Science:

    • Cell Biology
    • Molecular Biology
    • Biochemistry

    Background:

    • HeLa cells are a widely used human cancer cell line for biological research.
    • DNA replication is a fundamental process for cell division and genetic integrity.

    Purpose of the Study:

    • To investigate DNA replication in permeabilized HeLa cells using exogenous nucleotides.
    • To determine if DNA synthesis could be initiated and sustained in vitro.

    Main Methods:

    • HeLa cells were treated with a hypotonic buffer to induce cell permeability.
    • Permeabilized cells in S-phase were supplied with deoxyriboside triphosphates and other necessary components.
    • DNA replication activity was measured at different cell cycle stages.

    Main Results:

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    • Permeabilized HeLa cells synthesized DNA when provided with exogenous nucleotides, ATP, Mg2+, and suitable ionic conditions.
    • Replication initiated at sites active before cell treatment and was confined to the nucleus.
    • Up to 5% of the HeLa genome could be replicated under optimal conditions.
    • DNA replicase activity in permeabilized cells correlated with [14C] thymidine incorporation in intact cells.

    Conclusions:

    • Hypotonic treatment provides a method to study DNA replication in HeLa cells with exogenous substrates.
    • This technique allows for the investigation of DNA replication machinery and regulation in vitro.
    • The results demonstrate the feasibility of replicating large portions of the genome using external nucleotide sources.