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The unstructured C-terminal tail of the 9-1-1 clamp subunit Ddc1 activates Mec1/ATR via two distinct mechanisms

Vasundhara M Navadgi-Patil1, Peter M Burgers

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

Molecular Cell
|December 17, 2009
PubMed

Insights

The 9-1-1 checkpoint clamp activates the Mec1/ATR protein kinase in response to DNA damage. Ddc1, a subunit of 9-1-1, directly activates Mec1 in G1 and G2, with Dpb11/TopBP1 involvement in G2.

Area of Science:

  • Cellular biology
  • Molecular genetics
  • Biochemistry

Background:

  • DNA damage checkpoint pathways are crucial for preventing cell-cycle progression under stress.
  • Mec1-Ddc2 (human ATR-ATRIP) is the key checkpoint kinase in S. cerevisiae.
  • The 9-1-1 checkpoint clamp and Dpb11/TopBP1 are known potential activators of Mec1/ATR.

Purpose of the Study:

  • To elucidate the specific roles of the 9-1-1 checkpoint clamp and Dpb11/TopBP1 in Mec1/ATR activation during different cell-cycle phases.
  • To identify the key domains and residues within Ddc1 responsible for Mec1 activation.

Main Methods:

  • Biochemical assays to study Mec1 activation in vitro.
  • Analysis of checkpoint function in G1 and G2 phases.
  • Site-directed mutagenesis to investigate the role of specific Ddc1 residues (W352, W544) and motifs.

Main Results:

  • Ddc1 activates Mec1 in G1 independently of Dpb11.
  • In G2, 9-1-1 activates Mec1 through both direct Ddc1 activation and Dpb11 recruitment via Ddc1 T602 phosphorylation.
  • Two conserved tryptophan residues (W352, W544) in Ddc1 are essential for Mec1 activation and checkpoint function.
  • Fused peptides containing these two motifs can activate Mec1.

Conclusions:

  • Ddc1 is a direct activator of Mec1/ATR kinase in both G1 and G2 phases.
  • Dpb11/TopBP1 acts as an adaptor in G2, facilitating Mec1 activation through Ddc1.
  • Specific aromatic residues within Ddc1 are critical for its function as a Mec1 activator, suggesting a conserved mechanism for checkpoint control.

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