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Related Experiment Videos

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

C D Lu1, J J Byrnes

  • 1Department of Medicine, University of Miami, Veterans Administration Medical Center, Florida 33125.

Biochemistry
|December 15, 1992
PubMed
Summary

Rabbit bone marrow contains proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta, which exhibits low processivity alone but increases significantly with PCNA. Some delta polymerase may be reclassified as epsilon.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Proliferating cell nuclear antigen (PCNA) is crucial for DNA replication.
  • DNA polymerase delta plays a key role in DNA repair and replication.
  • Understanding the characteristics of DNA polymerase delta and its interaction with PCNA is essential for comprehending DNA metabolism.

Purpose of the Study:

  • To partially purify and characterize PCNA and PCNA-dependent DNA polymerase delta from rabbit bone marrow.
  • To compare the properties of rabbit DNA polymerase delta with those of calf thymus DNA polymerase delta.
  • To investigate the processivity of DNA polymerase delta and the effect of PCNA on its activity.

Main Methods:

  • Glycerol density gradient centrifugation to determine sedimentation coefficient.

Related Experiment Videos

  • Western blotting using polyclonal antibodies to calf thymus PCNA-dependent DNA polymerase delta.
  • Enzyme activity assays to assess DNA polymerase and exonuclease activity.
  • Processivity assays with and without PCNA.

    • Main Results:

    • Rabbit DNA polymerase delta sediments at 8.2 S and shows a 125-123-kDa doublet and 48-kDa polypeptides correlating with activity.
    • The enzyme is recognized by antibodies to calf thymus PCNA-dependent DNA polymerase delta.
    • A 3'-->5' exonuclease copurifies with the DNA polymerase.
    • DNA polymerase delta exhibits low processivity alone, which is significantly enhanced by PCNA.
    • A previously described rabbit bone marrow DNA polymerase delta, lacking PCNA dependence and antibody recognition, is likely DNA polymerase epsilon.

    Conclusions:

    • Rabbit bone marrow contains a PCNA-dependent DNA polymerase delta with properties similar to its calf thymus counterpart.
    • PCNA significantly enhances the processivity of DNA polymerase delta.
    • A distinct DNA polymerase delta (epsilon) exists in rabbit bone marrow, characterized by high processivity and lack of PCNA dependence.
    • PCNA-dependent DNA polymerase delta constitutes a minor fraction of the total DNA polymerase activity in rabbit bone marrow.