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Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase

B L Pike1, A Hammet, J Heierhorst

  • 1St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.

The Journal of Biological Chemistry
|March 30, 2001
PubMed

Insights

The yeast Rad53 kinase

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • DNA Damage Response

Background:

  • Forkhead-associated (FHA) domains are critical phosphopeptide-binding modules.
  • Rad53-like kinases, including human Chk2, are vital for cell cycle arrest and DNA repair.
  • These kinases respond to DNA damage and replication stress.

Purpose of the Study:

  • To investigate the specific role of the N-terminal FHA domain (FHA1) of yeast Rad53 in cell cycle control.
  • To determine if FHA1 is essential for Rad53's checkpoint function.

Main Methods:

  • Ectopic expression of Rad53-FHA1 domain in yeast.
  • Site-directed mutagenesis to disrupt phosphopeptide binding.
  • Assessing cell cycle arrest and DNA damage sensitivity phenotypes.
  • Allelic replacement of the RAD53 gene.

Main Results:

  • Ectopic expression of Rad53-FHA1 caused a G(1) cell cycle arrest.
  • This phenotype was specific to Rad53-FHA1 and dependent on phosphopeptide binding.
  • Mutations in FHA1 led to increased DNA damage sensitivity in vivo.
  • Rad53-FHA1 domain is crucial for checkpoint function.

Conclusions:

  • The FHA1 domain of Rad53 is essential for proper checkpoint control.
  • FHA1 likely functions by binding to a phosphorylated target protein during G(1) phase.
  • This interaction is critical for responding to DNA damage.

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