Monitoring ATM kinase activity in living cells

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• DNA double-strand breaks (DSB) activate ATM protein kinase in mammalian cells.
• ATM activation initiates signaling cascades for DNA repair, cell cycle arrest, or apoptosis.
• Understanding ATM kinase dynamics is crucial for comprehending the DNA damage response.

Purpose of the Study:

• To develop a novel biosensor for measuring ATM kinase activity in real-time within single living cells.
• To investigate the spatiotemporal dynamics of ATM activation in response to DNA damage.
• To provide a tool for deeper insights into the DNA damage response pathways.

Main Methods:

• Development of a Förster Resonance Energy Transfer (FRET)-based biosensor using CFP and YFP fluorescent proteins.
• The biosensor incorporates an ATM phosphorylation site and an FHA phosphospecific binding domain.
• Measurement of FRET efficiency changes indicative of ATM kinase activity upon phosphorylation.

Main Results:

• The developed biosensor successfully measures ATM kinase activity in response to DSBs in living cells.
• The biosensor demonstrates specificity for ATM kinase, distinguishing it from ATR or DNA-PK.
• The reporter enables visualization of the spatiotemporal dynamics of ATM activity.

Conclusions:

• The novel FRET-based biosensor is a valuable tool for quantifying ATM kinase activity in living cells.
• This reporter facilitates a more detailed understanding of the DNA damage response and ATM signaling.
• The findings pave the way for further research into cellular responses to DNA damage.