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Published on: June 9, 2017
Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM
Yi Zhou1, Ji-Hoon Lee1, Wenxia Jiang2
1Howard Hughes Medical Institute, University of Texas at Austin, Austin, TX 78712, USA; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Abstract:
Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.
Insights
DNA-dependent protein kinase (DNA-PKcs) negatively regulates Ataxia-telangiectasia mutated (ATM) through phosphorylation. This discovery reveals a key mechanism controlling DNA repair pathway choice and ATM signaling.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- Ataxia-telangiectasia mutated (ATM) is crucial for DNA damage response and repair via homologous recombination.
- Understanding ATM regulation is vital for comprehending DNA repair fidelity and cellular survival.
Purpose of the Study:
- To investigate the regulatory relationship between ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs).
- To elucidate the mechanism by which DNA-PKcs influences ATM activity and DNA repair pathway selection.
Main Methods:
- Utilized chemical inhibition and gene deletion of DNA-PKcs in human cells.
- Performed in vitro biochemical assays with purified ATM and DNA-PKcs proteins.
- Characterized ATM phosphorylation sites using phospho-mimetic and phospho-blocking mutations.
Main Results:
- ATM exhibits hyperactivity upon DNA-PKcs inhibition or deletion.
- Active DNA-PKcs directly reduces ATM kinase activity in vitro.
- Phosphorylation of ATM by DNA-PKcs at specific sites inhibits ATM activity and signaling.
- Blocking these phosphorylation sites increases apoptosis frequency.
Conclusions:
- DNA-PKcs acts as a negative regulator of ATM activity through direct phosphorylation.
- This regulatory axis plays a significant role in controlling DNA repair pathway choice.
- The findings provide novel insights into the intricate network governing DNA damage response.
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