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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

Purification of the DNA-PK/PARP1 complex. (A) Schematics of purification protocol. (B) Silver stained SDS–PAGE analysis of the purified complex, showing four apparently stoichiometric bands for the DNA-PKcs, PARP1, Ku70 and Ku80 polypeptides. Electron microscopy and single particle analysis of the DNA-PK/PARP1 complex. (C) Raw micrograph, showing heterogeneity in the sample (monomeric and dimeric complexes coexist, in analogy with the DNA-PK complex in isolation). White circles: monomers; white squares: dimers. (D and E) Reference-free class averages of monomeric complex show heterogeneity, since related views can either have or not have a strong density (indicated by the white arrow in (E). (F) Class averages used in the final 3D reconstruction. (G) Reprojections of the 3D model. (H) 3D model calculated by single particle analysis, accounting for the volume of the sum of DNA-PKcs, Ku heterodimers and PARP1. I. Fourier Shell Correlation plot.
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gkr1231-F2: Purification of the DNA-PK/PARP1 complex. (A) Schematics of purification protocol. (B) Silver stained SDS–PAGE analysis of the purified complex, showing four apparently stoichiometric bands for the DNA-PKcs, PARP1, Ku70 and Ku80 polypeptides. Electron microscopy and single particle analysis of the DNA-PK/PARP1 complex. (C) Raw micrograph, showing heterogeneity in the sample (monomeric and dimeric complexes coexist, in analogy with the DNA-PK complex in isolation). White circles: monomers; white squares: dimers. (D and E) Reference-free class averages of monomeric complex show heterogeneity, since related views can either have or not have a strong density (indicated by the white arrow in (E). (F) Class averages used in the final 3D reconstruction. (G) Reprojections of the 3D model. (H) 3D model calculated by single particle analysis, accounting for the volume of the sum of DNA-PKcs, Ku heterodimers and PARP1. I. Fourier Shell Correlation plot.

Mentions: The samples were then loaded on a 18–60% glycerol gradient, with the buffer system 20 mM HEPES (pH 7.5), NaCl 200 mM, 1 mM DTT, 0.5 mM EDTA, 0.001% β-octylglucoside. Further separation was achieved by using a Beckman SW28 rotor, spinning for 72 h at 25 000 rpm. Fractions were collected from the bottom and analysed by means of SDS–PAGE on 4–12% Bis-Tris NOVEX gradient gels (Invitrogen, UK). This analysis highlighted the co-migration of stoichiometric quantities of an additional protein with the three protein components of DNA-PK (Figure 2B). The fourth protein species was identified as PARP1 by mass spectrometry and immunoblotting (data not shown).


Visualization of a DNA-PK/PARP1 complex.

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

Purification of the DNA-PK/PARP1 complex. (A) Schematics of purification protocol. (B) Silver stained SDS–PAGE analysis of the purified complex, showing four apparently stoichiometric bands for the DNA-PKcs, PARP1, Ku70 and Ku80 polypeptides. Electron microscopy and single particle analysis of the DNA-PK/PARP1 complex. (C) Raw micrograph, showing heterogeneity in the sample (monomeric and dimeric complexes coexist, in analogy with the DNA-PK complex in isolation). White circles: monomers; white squares: dimers. (D and E) Reference-free class averages of monomeric complex show heterogeneity, since related views can either have or not have a strong density (indicated by the white arrow in (E). (F) Class averages used in the final 3D reconstruction. (G) Reprojections of the 3D model. (H) 3D model calculated by single particle analysis, accounting for the volume of the sum of DNA-PKcs, Ku heterodimers and PARP1. I. Fourier Shell Correlation plot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1231-F2: Purification of the DNA-PK/PARP1 complex. (A) Schematics of purification protocol. (B) Silver stained SDS–PAGE analysis of the purified complex, showing four apparently stoichiometric bands for the DNA-PKcs, PARP1, Ku70 and Ku80 polypeptides. Electron microscopy and single particle analysis of the DNA-PK/PARP1 complex. (C) Raw micrograph, showing heterogeneity in the sample (monomeric and dimeric complexes coexist, in analogy with the DNA-PK complex in isolation). White circles: monomers; white squares: dimers. (D and E) Reference-free class averages of monomeric complex show heterogeneity, since related views can either have or not have a strong density (indicated by the white arrow in (E). (F) Class averages used in the final 3D reconstruction. (G) Reprojections of the 3D model. (H) 3D model calculated by single particle analysis, accounting for the volume of the sum of DNA-PKcs, Ku heterodimers and PARP1. I. Fourier Shell Correlation plot.
Mentions: The samples were then loaded on a 18–60% glycerol gradient, with the buffer system 20 mM HEPES (pH 7.5), NaCl 200 mM, 1 mM DTT, 0.5 mM EDTA, 0.001% β-octylglucoside. Further separation was achieved by using a Beckman SW28 rotor, spinning for 72 h at 25 000 rpm. Fractions were collected from the bottom and analysed by means of SDS–PAGE on 4–12% Bis-Tris NOVEX gradient gels (Invitrogen, UK). This analysis highlighted the co-migration of stoichiometric quantities of an additional protein with the three protein components of DNA-PK (Figure 2B). The fourth protein species was identified as PARP1 by mass spectrometry and immunoblotting (data not shown).

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