Budding yeast Rad9 is an ATP-dependent Rad53 activating machine

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.
• 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.