DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Excessive DNA damage can cause mitochondrial dysfunction, reduced energy production, and increased reactive oxygen species (ROS).
• The ataxia telangiectasia mutant (ATM) protein is a key regulator of cellular responses to both DNA damage and mitochondrial dysfunction.
• Distinguishing ATM activation by DNA damage versus mitochondrial dysfunction has been challenging.

Purpose of the Study:

• To determine if nuclear and/or mitochondrial DNA double-strand breaks (DSBs) can activate ATM independently of mitochondrial dysfunction.
• To investigate the localization of activated ATM and its substrates following DNA damage at different cellular compartments.

Main Methods:

• Generated nuclear and mitochondrial DNA DSBs in A549 lung adenocarcinoma cells using retroviruses expressing PstI endonuclease fused to nuclear localization sequence (NLS) or mitochondrial targeting sequence (MTS).
• Assessed DNA damage response by measuring phosphorylation of ATM, p53 (TP53), KAP1, and SMC1.
• Analyzed protein localization in nuclear and mitochondrial fractions and measured p21 expression and cell growth.

Main Results:

• Expression of MTS-PstI-HA and NLS-PstI-HA induced DNA damage response, including ATM phosphorylation, independent of detectable mitochondrial dysfunction or excess ROS.
• Phosphorylated ATM and SMC1 were primarily nuclear, while phosphorylated TP53 and KAP1 were found in both nuclear and mitochondrial fractions.
• PstI expression increased p21 levels and inhibited cell growth.

Conclusions:

• DNA double-strand breaks (DSBs) are sufficient to activate ATM independently of mitochondrial dysfunction.
• The activated ATM protein and some substrates appear to be restricted to the nuclear compartment, irrespective of the DNA damage site.
• This suggests a compartmentalized signaling response to DNA damage.