Quantitative proteomics reveal ATM kinase-dependent exchange in DNA damage response complexes

Area of Science:

• Cellular Biology
• Molecular Oncology
• Radiation Biology

Background:

• ATM (Ataxia-Telangiectasia Mutated) is a key protein kinase in the DNA damage response (DDR) pathway activated by ionizing radiation (IR).
• The precise role of ATM in orchestrating protein interactions and dynamics within DDR complexes remains incompletely understood.

Purpose of the Study:

• To investigate the impact of ATM kinase activity on protein dynamics within DDR complexes using a specific inhibitor.
• To identify novel proteins involved in ATM-mediated DNA damage signaling and cellular response to radiation.

Main Methods:

• Utilized SILAC-based tandem mass spectrometry and subcellular fractionation to analyze protein changes in irradiated cells with and without the ATM inhibitor KU55933.
• Employed integrative network analysis to identify chromatin-enriched proteins interacting with known DNA repair factors and modulated by KU55933.
• Assessed protein exchange dynamics using fluorescence recovery after photobleaching (FRAP) and evaluated cellular radiosensitivity following ANXA1 knockdown.

Main Results:

• Identified 53BP1 and annexin A1 (ANXA1) as key candidates modulated by ATM inhibition.
• Demonstrated that ATM inhibition reduces the exchange rate of GFP-53BP1 within DDR complexes.
• Showed that ANXA1 knockdown increases cellular sensitivity to IR, suggesting a radioprotective role independent of ATM kinase activity.

Conclusions:

• ATM kinase activity is crucial for regulating the dynamic exchange of proteins within DNA damage response complexes.
• Annexin A1 (ANXA1) plays a significant role in cellular radioprotection through an ATM-independent mechanism.