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Maintenance of the DNA-damage checkpoint requires DNA-damage-induced mediator protein oligomerization

Takehiko Usui1, Steven S Foster, John H J Petrini

  • 1Laboratory of Chromosome Biology, Sloan-Kettering Institute, New York, NY 10065, USA.

Molecular Cell
|February 4, 2009
PubMed

Insights

DNA damage triggers Rad9 protein oligomerization through its BRCT domain interaction with its phosphorylated SCD. This oligomerization is crucial for DNA damage checkpoint signaling and is regulated by a feedback loop involving Rad53 kinase.

Area of Science:

  • Molecular biology
  • Cellular biology
  • Biochemistry

Background:

  • Brca1 C-terminal (BRCT) domain proteins mediate cellular responses to DNA damage.
  • Oligomeric assembly of these proteins at DNA damage sites is observed, but its regulation and function are unclear.
  • Understanding these mechanisms is vital for comprehending DNA repair and genomic stability.

Purpose of the Study:

  • To elucidate the molecular mechanism of DNA-damage-induced oligomerization of the Saccharomyces cerevisiae BRCT protein, Rad9.
  • To investigate the role of Rad9 oligomerization in DNA damage checkpoint signaling and its regulation.
  • To identify the key protein interactions and post-translational modifications involved in Rad9 assembly.

Main Methods:

  • Site-directed mutagenesis to disrupt specific protein-protein interactions and phosphorylation sites.
  • Biochemical assays to assess protein oligomerization and kinase activity.
  • Yeast genetics and microscopy to evaluate DNA damage checkpoint function and Rad9 localization.

Main Results:

  • Rad9 oligomerization is mediated by the interaction between its tandem BRCT domain and its Mec1/Tel1-phosphorylated SQ/TQ cluster domain (SCD).
  • Impaired Rad9 oligomerization disrupts checkpoint maintenance, although Rad53 activation remains unaffected.
  • Rad53 phosphorylates the Rad9 BRCT domain, attenuating the BRCT-SCD interaction, and failure to do so leads to persistent Rad9 foci.

Conclusions:

  • Rad9 oligomerization is essential for sustaining DNA damage checkpoint signaling.
  • A feedback loop involving Rad53 phosphorylation of Rad9 regulates Rad9 oligomerization and tunes the DNA damage response.
  • This study reveals a novel regulatory mechanism for DNA-damage-induced protein assembly and checkpoint control.

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