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Updated: May 26, 2026
Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications
Published on: April 21, 2023
Visualization of a DNA-PK/PARP1 complex
Laura Spagnolo1, Jody Barbeau, Nicola J Curtin
1Cancer Research UK DNA Repair Enzymes Group, The Institute of Cancer Research, London SW3 6JB, UK. laura.spagnolo@ed.ac.uk
Abstract:
The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions.
Insights
DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are essential for rapid DNA repair. Their interaction and conformational changes in DNA-PK dimers are crucial for double-strand break repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are key enzymes involved in DNA damage repair.
- Both enzymes are activated by DNA strand breaks and have known interactions.
Purpose of the Study:
- To provide in vivo evidence for the necessity of DNA-PK and PARP1 in rapid DNA repair.
- To elucidate the structural and functional relationship between DNA-PK and PARP1 during DNA repair.
Main Methods:
- Purification of a DNA-PK/PARP1 complex loaded on DNA.
- Electron microscopy and single particle analysis of the complex.
- Comparison with DNA-PK holoenzyme and fitting of crystallographic structures.
Main Results:
- DNA-PK and PARP1 were found to be equally necessary for rapid DNA repair.
- PARP1 density was observed in close contact with the Ku subunit of DNA-PK.
- PARP1 binding induced significant conformational changes in the DNA-PK synaptic dimer assembly.
Conclusions:
- The study supports a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair.
- A Non-Homologous End Joining (NHEJ) model is proposed where protein-protein interactions alter DNA-PK dimer architecture at DSBs.
