Related Experiment Video
Updated: Aug 29, 2025
Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells
Published on: September 5, 2017
Substrate spectrum of PPM1D in the cellular response to DNA double-strand breaks
Justus F Gräf1, Ivan Mikicic1, Xiaofei Ping1,2,3
1Institute of Molecular Biology (IMB), 55128 Mainz, Germany.
Abstract:
PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing. One-third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder, and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.
Insights
Protein phosphatase PPM1D regulates the DNA damage response (DDR). This study identified 73 PPM1D substrates involved in DNA repair and RNA processing, revealing its role in modulating DNA double-strand break signaling.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- PPM1D is a p53-regulated protein phosphatase crucial for the DNA damage response (DDR).
- Alterations in PPM1D are common in various cancers.
- Understanding PPM1D's substrates is key to deciphering its role in DDR and cancer.
Purpose of the Study:
- To identify substrates of PPM1D following DNA double-strand breaks (DSBs).
- To investigate the role of PPM1D in counteracting ATM/ATR/DNA-PK signaling pathways.
- To characterize the sequence motifs targeted by PPM1D.
Main Methods:
- Chemical inhibition of PPM1D.
- Quantitative mass spectrometry-based phosphoproteomics.
- Etoposide treatment to induce DSBs.
Main Results:
- Identified 73 putative PPM1D substrates involved in DNA repair, transcription, and RNA processing.
- PPM1D dephosphorylates one-third of DSB-induced S/TQ phosphorylation sites, indicating partial counteraction of ATM/ATR/DNA-PK signaling.
- Discovered Kanadaptin as a novel ATM and PPM1D substrate.
- PPM1D-targeted phosphorylation sites are enriched in intrinsically disordered regions with specific sequence features.
Conclusions:
- PPM1D plays a significant role in the response to DSBs by regulating phosphorylation of DNA- and RNA-binding proteins.
- PPM1D's activity is partially antagonistic to major DDR kinases.
- The findings provide insights into PPM1D's function in maintaining genomic stability and its implications in cancer.
Related Concept Videos
02:04Fixing Double-strand Breaks
02:37DNA Damage can Stall the Cell Cycle
01:02Long-patch Base Excision Repair
01:54Base Excision Repair
The first step of...
02:31Homologous Recombination
01:38Nucleotide Excision Repair
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...