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Updated: Jun 6, 2026
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage
Ariel Bensimon1, Alexander Schmidt, Yael Ziv
1David and Inez Myers Laboratory for Cancer Genetics, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Abstract:
The double-strand break (DSB) is a cytotoxic DNA lesion caused by oxygen radicals, ionizing radiation, and radiomimetic chemicals. Cells cope with DNA damage by activating the DNA damage response (DDR), which leads either to damage repair and cellular survival or to programmed cell death. The main transducer of the DSB response is the nuclear protein kinase ataxia telangiectasia mutated (ATM). We applied label-free quantitative mass spectrometry to follow the dynamics of DSB-induced phosphoproteome in nuclear fractions of the human melanoma G361 cells after radiomimetic treatment. We found that these dynamics are complex, including both phosphorylation and dephosphorylation events. In addition to identifying previously unknown ATM-dependent phosphorylation and dephosphorylation events, we found that about 40% of DSB-induced phosphorylations were ATM-independent and that several other kinases are potentially involved. Sustained activity of ATM was required to maintain many ATM-dependent phosphorylations. We identified an ATM-dependent phosphorylation site on ATM itself that played a role in its retention on damaged chromatin. By connecting many of the phosphorylated and dephosphorylated proteins into functional networks, we highlight putative cross talks between proteins pertaining to several cellular biological processes. Our study expands the DDR phosphorylation landscape and identifies previously unknown ATM-dependent and -independent branches. It reveals insights into the breadth and complexity of the cellular responses involved in the coordination of many DDR pathways, which is in line with the critical importance of genomic stability in maintenance of cellular homeostasis.
Insights
DNA double-strand breaks trigger complex cellular responses. This study reveals new ATM-dependent and independent phosphorylation events, expanding our understanding of the DNA damage response (DDR) and genomic stability.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- DNA double-strand breaks (DSBs) are severe DNA lesions that activate the DNA damage response (DDR).
- The ataxia telangiectasia mutated (ATM) kinase is a central transducer of the DSB response, orchestrating repair and cell fate.
- Understanding the dynamic phosphoproteome following DSBs is crucial for comprehending cellular homeostasis and genomic stability.
Purpose of the Study:
- To investigate the dynamic phosphoproteome changes in human melanoma cells following DSB induction.
- To identify novel ATM-dependent and ATM-independent phosphorylation events in the DDR.
- To elucidate the role of ATM activity and its regulation in response to DNA damage.
Main Methods:
- Label-free quantitative mass spectrometry was employed to analyze nuclear phosphoproteome dynamics.
- Human melanoma G361 cells were treated with radiomimetic agents to induce DSBs.
- Bioinformatic analysis was used to construct functional networks of phosphorylated and dephosphorylated proteins.
Main Results:
- DSB induction resulted in complex phosphorylation and dephosphorylation dynamics.
- Numerous novel ATM-dependent and ATM-independent phosphorylation events were identified.
- Approximately 40% of DSB-induced phosphorylations were found to be ATM-independent, suggesting involvement of other kinases.
- An ATM-dependent phosphorylation site on ATM itself was identified, crucial for its chromatin retention.
- Functional networks revealed cross-talk between proteins involved in various cellular processes.
Conclusions:
- The study expands the known landscape of DDR-induced phosphorylation.
- It highlights the existence of distinct ATM-dependent and -independent DDR pathways.
- The findings provide insights into the complexity of cellular responses coordinating DDR pathways for genomic stability.
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