DNA double strand breaks (DSB) in mammalian cells result in the activation of the ATM protein kinase. This leads to phosphorylation of numerous downstream transducer and effector proteins that coordinate a cellular response including DNA repair, cell cycle arrest or apoptosis. We have developed a reporter protein that allows the measurement of ATM kinase activity in single living cells. This CFP-YFP FRET-based biosensor uses an ATM phosphorylation site and an FHA phosphospecific binding domain to produce a phosphorylation-induced change in conformation, which alters the FRET efficiency between CFP and YFP. We show that the reporter provides a measurable output in response to DSBs and is specific for ATM over ATR or DNA-PK. We expect the description of the spatiotemporal dynamics of ATM activity in living cells that this reporter provides will be helpful in providing a more detailed understanding of the DNA damage response.
|Number of pages||8|
|State||Published - Sep 1 2007|