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Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase

Vasundhara M Navadgi-Patil1, Peter M Burgers

  • 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The Journal of Biological Chemistry
|October 17, 2008
PubMed

Insights

The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase, also known as ATR-ATRIP, is activated by Dpb11 and the 9-1-1 complex. These activators synergize to promote cell cycle arrest in response to DNA damage.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase (ATR-ATRIP) is crucial for DNA damage and replication stress response.
  • The DNA damage checkpoint clamp Ddc1-Mec3-Rad17 (9-1-1) and Dpb11 (TopBP1) are involved in checkpoint activation.
  • Dpb11 is essential for DNA replication initiation and checkpoint control.

Purpose of the Study:

  • To investigate the role of Dpb11 in Mec1 kinase activation.
  • To determine if DNA is required for Dpb11's function as an activator.
  • To explore the synergistic activation of Mec1 by Dpb11 and the 9-1-1 complex.

Main Methods:

  • In vitro kinase assays to assess Mec1 activation.
  • Phosphorylation assays of downstream targets like Rad53 and RPA.
  • Comparative analysis of Dpb11 and 9-1-1 activation, both independently and in combination.

Main Results:

  • Dpb11 directly activates Mec1 kinase, leading to phosphorylation of Rad53 and DNA-bound RPA.
  • DNA is not essential for Dpb11 to function as a Mec1 activator.
  • Dpb11 and the 9-1-1 complex show independent activation of Mec1, with significant synergy when both are present.

Conclusions:

  • Dpb11 is a direct activator of Mec1 kinase.
  • Dpb11 and the 9-1-1 complex may partially compensate for each other in yeast checkpoint function.
  • The combined action of Dpb11 and 9-1-1 provides a robust mechanism for Mec1 activation during cellular stress.

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