Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment

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Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
10:24

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Published on: September 28, 2012

Boris Pfander1, John F X Diffley

  • 1Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.

The EMBO Journal
|September 28, 2011
PubMed

Insights

The Saccharomyces cerevisiae Dpb11 protein coordinates DNA damage checkpoint signaling. A Dpb11-Mec1-Rad9 complex is essential for Rad9 phosphorylation and checkpoint activation, ensuring timely cellular response.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Eukaryotic cells activate DNA damage checkpoint signaling pathways in response to genetic damage.
  • These pathways involve protein kinase cascades and non-kinase mediator proteins.
  • Saccharomyces cerevisiae Dpb11 is a key mediator that activates the Mec1 kinase.

Purpose of the Study:

  • To investigate the role of a ternary complex involving Dpb11, Mec1, and Rad9 in DNA damage checkpoint activation.
  • To elucidate the mechanism of Rad9 phosphorylation and recruitment by Mec1 and Dpb11.
  • To understand the temporal and spatial regulation of Dpb11-mediated checkpoint signaling.

Main Methods:

  • In vitro biochemical assays to study protein interactions and kinase activity.
  • In vivo experiments to assess checkpoint activation in Saccharomyces cerevisiae.
  • Analysis of protein phosphorylation sites and their role in complex formation.

Main Results:

  • A ternary complex of Dpb11, Mec1, and Rad9 is crucial for efficient Rad9 phosphorylation by Mec1 in vitro and checkpoint activation in vivo.
  • Cyclin-dependent kinase (CDK) phosphorylation of Rad9 creates a binding site for Dpb11's BRCT repeats, facilitating Rad9 recruitment.
  • Checkpoint signaling through Dpb11 is inefficient during the G1 phase due to inactive CDK.

Conclusions:

  • Dpb11 coordinates checkpoint signal transduction both temporally and spatially.
  • Ensures the initiator kinase Mec1 is activated specifically near its critical substrate Rad9.
  • This mechanism highlights the precise regulation of DNA damage response pathways.

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