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Activation of topoisomerase I by poly [ADP-ribose] polymerase

P I Bauer1, K G Buki, J A Comstock

  • 1Octamer, Inc., San Leandro, CA 94710, USA.

International Journal of Molecular Medicine
|April 14, 2000
PubMed

Insights

Poly(ADP-ribose) polymerase (PARP I) significantly enhances Topoisomerase I (Topo I) activity by binding to DNA. This interaction increases Topo I

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) are crucial enzymes involved in DNA repair and replication.
  • Previous studies suggested potential interactions between PARP I and Topo I, but the precise nature of their relationship remained unclear.
  • Understanding enzyme interactions is vital for elucidating complex cellular processes like DNA metabolism.

Purpose of the Study:

  • To investigate the functional interaction between purified Poly(ADP-ribose) polymerase I (PARP I) and Topoisomerase I (Topo I).
  • To determine the molecular mechanisms by which PARP I influences Topo I activity and DNA binding.
  • To explore the role of PARP I in modulating Topo I's catalytic cycle.

Main Methods:

  • Re-isolation and purification of PARP I and Topo I from calf thymus to ensure high purity.
  • Enzyme activity assays to measure Topo I catalytic rates in the presence and absence of PARP I.
  • Kinetic analysis, including binding constant determination and computer modeling.
  • Fluorescence resonance energy transfer (FRET) to study protein-protein interactions.
  • DNA binding assays using circular SV40 DNA to quantify Topo I-DNA complex formation.

Main Results:

  • PARP I significantly increased the specific activity of Topo I in a saturation-dependent manner.
  • Recombinant and purified PARP I exhibited identical activation of Topo I, confirming the observed effect.
  • Binding constants supported a model where PARP I-Topo I association is rate-limiting for Topo I activation.
  • Specific domains of PARP I involved in protein-DNA and protein-protein interactions were found to be essential for Topo I activation.
  • PARP I binding to SV40 DNA enhanced Topo I's association with the DNA, potentially by increasing DNA superhelicity.

Conclusions:

  • PARP I directly and kinetically activates Topo I, suggesting a synergistic role in DNA processing.
  • The interaction between PARP I and Topo I is mediated by specific protein domains and influences Topo I's DNA binding.
  • PARP I may enhance Topo I's catalytic efficiency by facilitating the formation of a more stable Topo I-DNA complex, impacting the DNA breakage-reunion cycle.

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