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DNA polymerase delta: one polypeptide, two activities.

L P Goscin, J J Byrnes

    Biochemistry
    |May 11, 1982
    PubMed
    Summary

    Rabbit bone marrow DNA polymerase delta, purified to homogeneity, exhibits both polymerase and exonuclease activity. Aphidicolin inhibits this enzyme, challenging its specificity for DNA polymerase alpha.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • DNA polymerase delta from rabbit bone marrow possesses a 3'-5'-exonuclease activity.
    • Previous characterization indicated a molecular weight of 122,000 and a frictional coefficient of 1.39.
    • Challenges in purification and analysis of DNA polymerase delta were previously encountered due to limited protein yield.

    Purpose of the Study:

    • To overcome purification challenges and achieve apparent homogeneity of DNA polymerase delta.
    • To further define the enzymatic properties and subunit composition of DNA polymerase delta.
    • To re-evaluate the specificity of aphidicolin as an inhibitor of DNA polymerase alpha.

    Main Methods:

    • Purification of DNA polymerase delta to apparent homogeneity.
    • Nondenaturing microgel electrophoresis and in situ activity assays.
    • Sodium dodecyl sulfate (SDS) gel electrophoresis for subunit analysis.
    • Enzymatic assays for DNA polymerase and exonuclease activity.
    • Inhibition studies using aphidicolin.

    Main Results:

    • DNA polymerase delta was purified to apparent homogeneity, revealing a single polypeptide of 122,000 apparent molecular weight on SDS-PAGE.
    • The purified enzyme exhibited both DNA polymerase activity, incorporating dTMP into poly(dA)/oligo(dT), and 3'-5'-exonuclease activity, hydrolyzing newly synthesized DNA.
    • Aphidicolin inhibited both the polymerase and exonuclease activities of DNA polymerase delta.
    • DNA polymerase alpha from the same source does not share subunits with DNA polymerase delta.

    Conclusions:

    • DNA polymerase delta is a distinct enzyme with associated exonuclease function.
    • Aphidicolin's inhibitory effect on DNA polymerase delta suggests it is not exclusively specific for DNA polymerase alpha.
    • Previous conclusions regarding aphidicolin's specificity for DNA polymerase alpha require reconsideration based on these findings.

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