Requirement of the prolyl isomerase Pin1 for the replication checkpoint

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Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells
08:38

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Published on: March 3, 2015

K E Winkler1, K I Swenson, S Kornbluth

  • 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710, USA.

Science (New York, N.Y.)
|March 4, 2000
PubMed

Insights

The peptidyl-prolyl isomerase Pin1 is crucial for the DNA replication checkpoint. Without Pin1, cells prematurely enter mitosis, indicating its role in delaying cell division until DNA replication is complete.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The peptidyl-prolyl isomerase Pin1 plays a role in cell cycle regulation.
  • Pin1's specific function in DNA replication checkpoints is not fully understood.

Purpose of the Study:

  • To investigate the role of Pin1 in the DNA replication checkpoint in Xenopus laevis egg extracts.
  • To determine if Pin1 is required to prevent premature mitotic entry during replication stress.

Main Methods:

  • Xenopus laevis egg extracts were depleted of Pin1.
  • DNA replication was inhibited using aphidicolin.
  • Recombinant wild-type Pin1 and an inactive mutant were added to assess rescue of checkpoint function.
  • Levels of Cdc25 phosphorylation, Cdc2/cyclin B activity, and MPM-2 epitopes were analyzed.

Main Results:

  • Pin1-depleted extracts entered mitosis prematurely when DNA replication was inhibited.
  • Addition of wild-type Pin1 restored the replication checkpoint, preventing premature mitotic entry.
  • An isomerase-inactive Pin1 mutant failed to rescue the checkpoint defect.
  • Premature mitosis was associated with Cdc25 hyperphosphorylation and Cdc2/cyclin B activation.

Conclusions:

  • Pin1 is essential for the DNA replication checkpoint in Xenopus.
  • Pin1 activity is required to delay the onset of mitosis in response to incomplete DNA replication.
  • Pin1 likely functions by regulating key cell cycle proteins like Cdc25.

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