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Budding yeast Rad9 is an ATP-dependent Rad53 activating machine.

C S Gilbert1, C M Green, N F Lowndes

  • 1Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South Mimms, EN6 3LD, Hertfordshire, United Kingdom.

Molecular Cell
|August 21, 2001
PubMed
Summary

Budding yeast Rad9 forms distinct complexes, with a DNA damage-induced complex activating Rad53 kinase. This Rad9-Rad53 complex acts as a scaffold, promoting Rad53 autophosphorylation and activation.

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Area of Science:

  • Cellular biology
  • Molecular mechanisms of DNA repair

Background:

  • Rad9 is a key protein in the DNA damage response pathway in budding yeast.
  • The post-translational modification and complex formation of Rad9 are critical for its function.

Purpose of the Study:

  • To investigate the distinct complexes of Rad9 in budding yeast.
  • To elucidate the role of Rad9 complex formation in the activation of Rad53 kinase following DNA damage.

Main Methods:

  • Analysis of cell extracts using size-exclusion chromatography.
  • Western blotting to detect phosphorylated and unphosphorylated forms of Rad9 and Rad53.
  • In vitro kinase assays to assess Rad53 activity.

Main Results:

  • Two distinct Rad9 complexes were identified: a larger (>850 kDa) hypophosphorylated form in non-damaged cells and a smaller (560 kDa) hyperphosphorylated form after DNA damage.

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  • The smaller complex contains hyperphosphorylated Rad9 and Rad53, and is capable of catalyzing Rad53 phosphorylation and activation.
  • Rad53 kinase activity is required for the formation and function of the smaller complex, while Mec1 and Tel1 kinases are not required once the complex is formed.

    • Conclusions:

    • DNA damage induces hyperphosphorylation of Rad9, leading to the formation of a smaller Rad9-Rad53 complex.
    • This complex functions as a scaffold to promote Rad53 in trans autophosphorylation and activation, independent of Mec1/Tel1 activity.
    • A model is proposed where Mec1/Tel1 initiate Rad9 hyperphosphorylation, facilitating Rad53 recruitment and subsequent self-activation via the Rad9 scaffold.