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Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg

Alessia Errico1, Vincenzo Costanzo, Tim Hunt

  • 1Clare Hall Laboratories, Cancer Research UK, South Mimms, Hertfordshire EN6 3LD, United Kingdom.

Proceedings of the National Academy of Sciences of the United States of America
|September 12, 2007
PubMed

Insights

The Tipin/Tim1 complex is crucial for DNA replication fork recovery and checkpoint activation after damage. Phosphorylation of Tipin by cyclin-dependent kinases regulates its function in these processes.

Area of Science:

  • Cellular biology
  • Molecular biology
  • DNA replication and repair

Background:

  • The Tipin/Tim1 complex is essential for DNA damage checkpoint response at replication forks.
  • Understanding the regulation and function of Tipin is key to comprehending DNA replication fidelity.

Purpose of the Study:

  • To investigate the role of Xenopus laevis Tipin in DNA replication and its regulation by cell cycle kinases.
  • To elucidate the function of the Tipin/Tim1 complex in replication fork stability and checkpoint activation.

Main Methods:

  • Identification of Tipin as a substrate for cyclin E/cyclin-dependent kinases 2.
  • Analysis of Tipin phosphorylation during interphase and mitosis.
  • Chromatin binding assays for the Tipin/Tim1 complex and its interaction with MCM helicase.
  • Xenopus extract depletion experiments to assess Tipin's role in replication recovery and checkpoint activation.

Main Results:

  • Xenopus Tipin is phosphorylated by cyclin E/CDK2 in interphase and further in mitosis.
  • The Tipin/Tim1 complex associates with chromatin and interacts with the MCM helicase during normal replication.
  • Tipin depletion impairs stalled replication fork recovery and Chk1 activation following aphidicolin treatment.
  • Failure to load Claspin onto chromatin was observed in Tipin-depleted extracts.

Conclusions:

  • Tipin phosphorylation by cyclin E/CDK2 is a regulatory mechanism for its function.
  • The Tipin/Tim1 complex is involved in maintaining replication fork stability and facilitating checkpoint activation.
  • Tipin plays a critical role in the recovery of stalled replication forks and the DNA damage response pathway.

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