Yeast PP4 interacts with ATR homolog Ddc2-Mec1 and regulates checkpoint signaling

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Mec1-Ddc2 (ATR-ATRIP) is a key regulator of the DNA damage checkpoint and cell-cycle progression in yeast.
• Understanding Mec1-Ddc2 regulation is crucial for comprehending DNA repair mechanisms.

Purpose of the Study:

• To identify novel regulators of the Mec1-Ddc2 complex.
• To elucidate the functional relationship between Mec1-Ddc2 and the PP4 phosphatase complex.

Main Methods:

• Utilized a mec1 mutant compromised in the intra-S phase checkpoint for genetic screens.
• Employed spontaneous survivor screens and E-MAP (Environmental المصفوف Protein Array) for synthetic lethality.
• Performed phosphoproteomic analysis and co-immunoprecipitation assays.
• Investigated physical interactions using Förster Resonance Energy Transfer (FRET) in subnuclear foci.

Main Results:

• Loss of PP4 phosphatase (pph3Δ) and its cofactor Psy2 (psy2Δ) were identified as potent suppressors of mec1-100 lethality.
• PP4 regulates 94% of Mec1-Ddc2 targets under hydroxyurea (HU) stress, including a critical site on Mec1.
• Physical interaction between Mec1 and PP4, mediated by Psy2 and Ddc2, was confirmed in vivo.
• PP4 contributes to Mec1-Ddc2 function beyond Rad53 phosphorylation, indicating a direct regulatory role.

Conclusions:

• The study establishes a physical and functional link between Mec1-Ddc2 and the PP4 phosphatase complex.
• This Mec1-PP4 unit plays a critical role in regulating the DNA damage checkpoint response.
• The findings reveal a novel layer of regulation for Mec1-Ddc2, impacting DNA repair and cell-cycle fidelity.