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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

The dynamic nature of DNA-PKcs in the formation of dimers, as assessed by electron microscopy and single particle analysis. (A) DNA-PK dimers on Y-shaped DNA, with head domains facing opposite. (B) DNA-PK/PARP1 dimers on Y-shaped DNA, with head domains in close contact. (C) Schematics of the relative arrangement of DNA-PK head domains in DNA-PK dimers and DNA-PK/PARP1 dimers, both loaded on Y-shaped DNA. DNA-PKcs is coloured yellow, apart from the head domain, which is coloured red. Ku is represented as a blue ring and PARP1 as a green oval.
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gkr1231-F4: The dynamic nature of DNA-PKcs in the formation of dimers, as assessed by electron microscopy and single particle analysis. (A) DNA-PK dimers on Y-shaped DNA, with head domains facing opposite. (B) DNA-PK/PARP1 dimers on Y-shaped DNA, with head domains in close contact. (C) Schematics of the relative arrangement of DNA-PK head domains in DNA-PK dimers and DNA-PK/PARP1 dimers, both loaded on Y-shaped DNA. DNA-PKcs is coloured yellow, apart from the head domain, which is coloured red. Ku is represented as a blue ring and PARP1 as a green oval.

Mentions: A total of 3125 dimeric appearances of the DNA-PK/PARP1 complex were manually extracted to a sub-data set. They were then analysed in 2D by alignment and classification, revealing a ‘T-shaped’ architecture. Each DNA-PK/PARP1 assembly within class averages of this dimer-of-heterotetramers retains the general features of the smaller DNA-PK/PARP1 particles, with a clear added density attached to the Ku end of the DNA-PK complex. Importantly, in contrast to DNA-PK dimers loaded on Y-shaped DNA (Figure 4A) and similarly to DNA-PK dimers loaded on hairpin DNA, one DNA-PKcs head domain is located in close contact with the DNA-PKcs arm domain within the DNA-PKcs opposite (Figure 4B). The orientation of the two halves of the complex is however different from the one of DNA-PK dimers on hairpin DNA (Figure 4C). This orientation is compatible with the ability to auto-phosphorylate in trans (47), since the two main DNA-PKcs autophosphorylation clusters have been mapped in the HEAT repeats region, forming the arm and palm domain of DNA-PKcs (64). The dimers-of-tetramers were analysed as a single stack of particles, which all seemed to contain the PARP1 density, since all the class averages calculated present a strong signal at the same position of the PARP1 density in the tetramer (white arrowhead in Figure 4B, while the black arrowhead points to the DNA-PKcs head domain).Figure 4.


Visualization of a DNA-PK/PARP1 complex.

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

The dynamic nature of DNA-PKcs in the formation of dimers, as assessed by electron microscopy and single particle analysis. (A) DNA-PK dimers on Y-shaped DNA, with head domains facing opposite. (B) DNA-PK/PARP1 dimers on Y-shaped DNA, with head domains in close contact. (C) Schematics of the relative arrangement of DNA-PK head domains in DNA-PK dimers and DNA-PK/PARP1 dimers, both loaded on Y-shaped DNA. DNA-PKcs is coloured yellow, apart from the head domain, which is coloured red. Ku is represented as a blue ring and PARP1 as a green oval.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1231-F4: The dynamic nature of DNA-PKcs in the formation of dimers, as assessed by electron microscopy and single particle analysis. (A) DNA-PK dimers on Y-shaped DNA, with head domains facing opposite. (B) DNA-PK/PARP1 dimers on Y-shaped DNA, with head domains in close contact. (C) Schematics of the relative arrangement of DNA-PK head domains in DNA-PK dimers and DNA-PK/PARP1 dimers, both loaded on Y-shaped DNA. DNA-PKcs is coloured yellow, apart from the head domain, which is coloured red. Ku is represented as a blue ring and PARP1 as a green oval.
Mentions: A total of 3125 dimeric appearances of the DNA-PK/PARP1 complex were manually extracted to a sub-data set. They were then analysed in 2D by alignment and classification, revealing a ‘T-shaped’ architecture. Each DNA-PK/PARP1 assembly within class averages of this dimer-of-heterotetramers retains the general features of the smaller DNA-PK/PARP1 particles, with a clear added density attached to the Ku end of the DNA-PK complex. Importantly, in contrast to DNA-PK dimers loaded on Y-shaped DNA (Figure 4A) and similarly to DNA-PK dimers loaded on hairpin DNA, one DNA-PKcs head domain is located in close contact with the DNA-PKcs arm domain within the DNA-PKcs opposite (Figure 4B). The orientation of the two halves of the complex is however different from the one of DNA-PK dimers on hairpin DNA (Figure 4C). This orientation is compatible with the ability to auto-phosphorylate in trans (47), since the two main DNA-PKcs autophosphorylation clusters have been mapped in the HEAT repeats region, forming the arm and palm domain of DNA-PKcs (64). The dimers-of-tetramers were analysed as a single stack of particles, which all seemed to contain the PARP1 density, since all the class averages calculated present a strong signal at the same position of the PARP1 density in the tetramer (white arrowhead in Figure 4B, while the black arrowhead points to the DNA-PKcs head domain).Figure 4.

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