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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 effect of DNA-PK and PARP inhibitors on DSB repair. The recovery from IR-induced DSBs in the presence, or absence, of the DNA-PK inhibitor NU7441 or the PARP1 inhibitor KU-0058684, or both, was measured with g2HAX assays. (A) The effect on NU7441 on DNA-PK proficient and deficient cells. (B) The effect of KU-0058648 on PARP1 proficient and deficient cells. (C) The effect of NU7441 on PARP1 deficient cells and KU-0058684 on DNA-PK deficient cells. (D) The combinatorial effect of DNA-PK and PARP1 inhibitors on DNA-PK proficient cells. Cells were exposed to 2 Gy ionizing radiation and allowed to recover. Data are the mean ± standard error of three independent experiments over a 24-h time course.
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gkr1231-F1: The effect of DNA-PK and PARP inhibitors on DSB repair. The recovery from IR-induced DSBs in the presence, or absence, of the DNA-PK inhibitor NU7441 or the PARP1 inhibitor KU-0058684, or both, was measured with g2HAX assays. (A) The effect on NU7441 on DNA-PK proficient and deficient cells. (B) The effect of KU-0058648 on PARP1 proficient and deficient cells. (C) The effect of NU7441 on PARP1 deficient cells and KU-0058684 on DNA-PK deficient cells. (D) The combinatorial effect of DNA-PK and PARP1 inhibitors on DNA-PK proficient cells. Cells were exposed to 2 Gy ionizing radiation and allowed to recover. Data are the mean ± standard error of three independent experiments over a 24-h time course.

Mentions: We studied the role of PARP1 in the cellular response to clinically relevant IR doses by measuring DNA DSB repair in DNA-PK+/+ (V3-YAC), DNA-PK−/− (V3), PARP1+/+ and PARP1−/− cells. After irradiation of PARP1 proficient cells, DNA DSBs were rapidly induced as determined by γH2AX focus formation after 2 Gy IR (Figure 1). In repair-competent cell lines and consistent with our previous data with these cells (19) and independent data (59), DSB resolution followed a biphasic pattern. There was a rapid resolution of DSBs during the first 2 h with 80 ± 24% and 71 ± 10% being repaired in PARP1+/+ and V3-YAC cells, respectively. However, PARP1−/− MEFs and DNA-PKcs deficient V3 cells followed a different repair kinetics, with a much slower rate such that only 27 ± 21% and 25 ± 9%, respectively, had been repaired.Figure 1.


Visualization of a DNA-PK/PARP1 complex.

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

The effect of DNA-PK and PARP inhibitors on DSB repair. The recovery from IR-induced DSBs in the presence, or absence, of the DNA-PK inhibitor NU7441 or the PARP1 inhibitor KU-0058684, or both, was measured with g2HAX assays. (A) The effect on NU7441 on DNA-PK proficient and deficient cells. (B) The effect of KU-0058648 on PARP1 proficient and deficient cells. (C) The effect of NU7441 on PARP1 deficient cells and KU-0058684 on DNA-PK deficient cells. (D) The combinatorial effect of DNA-PK and PARP1 inhibitors on DNA-PK proficient cells. Cells were exposed to 2 Gy ionizing radiation and allowed to recover. Data are the mean ± standard error of three independent experiments over a 24-h time course.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1231-F1: The effect of DNA-PK and PARP inhibitors on DSB repair. The recovery from IR-induced DSBs in the presence, or absence, of the DNA-PK inhibitor NU7441 or the PARP1 inhibitor KU-0058684, or both, was measured with g2HAX assays. (A) The effect on NU7441 on DNA-PK proficient and deficient cells. (B) The effect of KU-0058648 on PARP1 proficient and deficient cells. (C) The effect of NU7441 on PARP1 deficient cells and KU-0058684 on DNA-PK deficient cells. (D) The combinatorial effect of DNA-PK and PARP1 inhibitors on DNA-PK proficient cells. Cells were exposed to 2 Gy ionizing radiation and allowed to recover. Data are the mean ± standard error of three independent experiments over a 24-h time course.
Mentions: We studied the role of PARP1 in the cellular response to clinically relevant IR doses by measuring DNA DSB repair in DNA-PK+/+ (V3-YAC), DNA-PK−/− (V3), PARP1+/+ and PARP1−/− cells. After irradiation of PARP1 proficient cells, DNA DSBs were rapidly induced as determined by γH2AX focus formation after 2 Gy IR (Figure 1). In repair-competent cell lines and consistent with our previous data with these cells (19) and independent data (59), DSB resolution followed a biphasic pattern. There was a rapid resolution of DSBs during the first 2 h with 80 ± 24% and 71 ± 10% being repaired in PARP1+/+ and V3-YAC cells, respectively. However, PARP1−/− MEFs and DNA-PKcs deficient V3 cells followed a different repair kinetics, with a much slower rate such that only 27 ± 21% and 25 ± 9%, respectively, had been repaired.Figure 1.

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