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Visualization of a DNA-PK/PARP1 complex.

Spagnolo L, Barbeau J, Curtin NJ, Morris EP, Pearl LH - Nucleic Acids Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer 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.

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Related in: MedlinePlus

Docking known X-ray structures into the DNA-PK/PARP1 density map. We used the UCSF Chimera software (58) to dock known structures for human DNA-PKcs (red), truncated human Ku heterodimer (green) and chicken PARP1 catalytic subunit (2PAW, blue) into the DNA-PK/PARP1 3D map.
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gkr1231-F3: Docking known X-ray structures into the DNA-PK/PARP1 density map. We used the UCSF Chimera software (58) to dock known structures for human DNA-PKcs (red), truncated human Ku heterodimer (green) and chicken PARP1 catalytic subunit (2PAW, blue) into the DNA-PK/PARP1 3D map.

Mentions: The resulting 3D map of the DNA-PK/PARP1 complex is ∼230 Å long, 160 Å wide and 120 Å deep. Within the map, regions can be readily recognized that are highly compatible with our previous analyses of DNA-PKcs, DNA-PK and Ku (6,27–29,63) as well as with the crystallographic structures of isolated components of the complex (35,64) (Figure 3). An extra density compatible with the size of full-length PARP1 can be visually located in contact with Ku (Figures 2H and 3). This observation is consistent with previous data showing genetic interaction between DNA-PK and PARP1 and the formation of a Ku/PARP1 complex (12–18), as well as for a physical role of PARP1 in modulating NHEJ.Figure 3.


Visualization of a DNA-PK/PARP1 complex.

Spagnolo L, Barbeau J, Curtin NJ, Morris EP, Pearl LH - Nucleic Acids Res. (2012)

Docking known X-ray structures into the DNA-PK/PARP1 density map. We used the UCSF Chimera software (58) to dock known structures for human DNA-PKcs (red), truncated human Ku heterodimer (green) and chicken PARP1 catalytic subunit (2PAW, blue) into the DNA-PK/PARP1 3D map.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351162&req=5

gkr1231-F3: Docking known X-ray structures into the DNA-PK/PARP1 density map. We used the UCSF Chimera software (58) to dock known structures for human DNA-PKcs (red), truncated human Ku heterodimer (green) and chicken PARP1 catalytic subunit (2PAW, blue) into the DNA-PK/PARP1 3D map.
Mentions: The resulting 3D map of the DNA-PK/PARP1 complex is ∼230 Å long, 160 Å wide and 120 Å deep. Within the map, regions can be readily recognized that are highly compatible with our previous analyses of DNA-PKcs, DNA-PK and Ku (6,27–29,63) as well as with the crystallographic structures of isolated components of the complex (35,64) (Figure 3). An extra density compatible with the size of full-length PARP1 can be visually located in contact with Ku (Figures 2H and 3). This observation is consistent with previous data showing genetic interaction between DNA-PK and PARP1 and the formation of a Ku/PARP1 complex (12–18), as well as for a physical role of PARP1 in modulating NHEJ.Figure 3.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer 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.

Show MeSH
Related in: MedlinePlus