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Repair replication in human cells. Simplified determination utilizing hydroxyurea.

C A Smith, P C Hanawalt

    Biochimica Et Biophysica Acta
    |May 19, 1976
    PubMed
    Summary
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    A simplified method determines DNA repair replication in mammalian cells using bromodeoxyuridine and radiolabels. This technique enhances accuracy for studying DNA repair synthesis in human cells.

    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • DNA repair replication is crucial for maintaining genomic integrity.
    • Accurate quantification of DNA repair synthesis is essential for understanding cellular responses to damage.
    • Existing methods for measuring DNA repair replication can be complex and time-consuming.

    Purpose of the Study:

    • To develop a simplified and shortened procedure for determining DNA repair replication in cultured mammalian cells.
    • To apply this method to normal diploid human cells (WI38) and their SV40 transformants (VA13).

    Main Methods:

    • Utilized bromodeoxyuridine (BrdU) as a density label and a radio-isotopic label for DNA.
    • Employed proteinase K digestion of lysed cells, followed immediately by alkaline cesium chloride density gradient centrifugation.

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  • Incorporated hydroxyurea to minimize semi-conservative DNA synthesis, enabling quantitative repair replication assessment via single centrifugation.

    • Main Results:

    • The developed procedure simplifies DNA repair replication measurement in human cells.
    • Hydroxyurea effectively reduces semi-conservative synthesis, allowing for quantitative repair analysis.
    • Repair synthesis in UV-irradiated cells increased by 25-40% with hydroxyurea when using thymidine as a tracer.
    • Hydroxyurea is not necessary under conditions of low semi-conservative synthesis (e.g., contact-inhibited cells, high UV doses).

    Conclusions:

    • The new method offers a streamlined approach to quantifying DNA repair replication.
    • This technique is applicable to both normal and transformed mammalian cells.
    • The findings provide a valuable tool for studying DNA repair mechanisms and responses to genotoxic agents.