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Related Experiment Videos

DNA synthesis in permeabilized mouse L cells.

N A Berger, E S Johnson

    Biochimica Et Biophysica Acta
    |February 18, 1976
    PubMed
    Summary

    Cold shock makes mouse L cells permeable to nucleotides, enabling DNA synthesis. This permeable cell system allows for in situ evaluation of DNA replication and polymerase activity.

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    Area of Science:

    • Cell Biology
    • Molecular Biology
    • Biochemistry

    Background:

    • Mouse L cells can be made permeable to nucleoside triphosphates using cold shock.
    • These permeable cells maintain structural integrity and can synthesize RNA and DNA from external nucleotide sources.

    Purpose of the Study:

    • To establish and validate a permeable cell system for studying DNA synthesis.
    • To investigate the requirements and inhibitors of DNA replication in situ.

    Main Methods:

    • Mouse L cells were treated with cold shock to induce permeability.
    • Exogenously supplied nucleotides and deoxynucleotides were used to assess DNA synthesis.
    • Thymidine kinase-deficient cells were employed to confirm direct deoxynucleotide incorporation.
    • Inhibitors such as N-ethylmaleimide, p-hydroxymercuribenzoate, actinomycin D, hydroxyurea, and cytosine arabinoside triphosphate were tested.

    Main Results:

    • Permeable mouse L cells synthesized nuclear DNA via semiconservative replication.
    • DNA synthesis required sodium ions (Na+), adenosine triphosphate (ATP), all four deoxynucleotides, and magnesium ions (Mg2+).
    • N-ethylmaleimide, p-hydroxymercuribenzoate, and actinomycin D inhibited DNA synthesis, while hydroxyurea and arabinosylcytosine did not.

    Conclusions:

    • The cold shock-induced permeable cell system is effective for studying DNA synthesis and replication.
    • This system allows for in situ analysis of replicative DNA polymerase activity using the cell's endogenous DNA template.
    • The findings provide insights into the mechanisms and regulation of DNA replication.

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