Visualization of a DNA-PK/PARP1 Complex

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2012 Jan 7

PMID 22223246

Citations 54

Authors

Laura Spagnolo

Jody Barbeau

Nicola J Curtin

Edward P Morris

Laurence H Pearl

Affiliations

Soon will be listed here.

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.

Citing Articles

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References

Cowell I, Durkacz B, Tilby M . Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA-dependent protein kinase and ataxia telangiectsia mutated. Biochem Pharmacol. 2005; 71(1-2):13-20. DOI: 10.1016/j.bcp.2005.09.029. View

Kawaichi M, Ueda K, HAYAISHI O . Multiple autopoly(ADP-ribosyl)ation of rat liver poly(ADP-ribose) synthetase. Mode of modification and properties of automodified synthetase. J Biol Chem. 1981; 256(18):9483-9. View

Leahy J, Golding B, Griffin R, Hardcastle I, Richardson C, Rigoreau L . Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett. 2004; 14(24):6083-7. DOI: 10.1016/j.bmcl.2004.09.060. View

Brewerton S, Dore A, Drake A, Leuther K, Blundell T . Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture. J Struct Biol. 2004; 145(3):295-306. DOI: 10.1016/j.jsb.2003.11.024. View

Jones B, Dale R . Radiobiologically based assessments of the net costs of fractionated focal radiotherapy. Int J Radiat Oncol Biol Phys. 1998; 41(5):1139-48. DOI: 10.1016/s0360-3016(98)00155-2. View

Dong F, Soubeyrand S, Hache R . Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5. Oncogene. 2010; 29(14):2093-103. DOI: 10.1038/onc.2009.492. View

Morrison C, Smith G, Stingl L, Jackson S, Wagner E, Wang Z . Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis. Nat Genet. 1997; 17(4):479-82. DOI: 10.1038/ng1297-479. View

Veuger S, Curtin N, Smith G, Durkacz B . Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair. Oncogene. 2004; 23(44):7322-9. DOI: 10.1038/sj.onc.1207984. View

Harris R, Esposito D, Sankar A, Maman J, Hinks J, Pearl L . The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR). J Mol Biol. 2003; 335(2):573-82. DOI: 10.1016/j.jmb.2003.10.047. View

10.

Mitchell J, Smith G, Curtin N . Poly(ADP-Ribose) polymerase-1 and DNA-dependent protein kinase have equivalent roles in double strand break repair following ionizing radiation. Int J Radiat Oncol Biol Phys. 2009; 75(5):1520-7. DOI: 10.1016/j.ijrobp.2009.07.1722. View

11.

Williams D, Lee K, Shi J, Chen D, Stewart P . Cryo-EM structure of the DNA-dependent protein kinase catalytic subunit at subnanometer resolution reveals alpha helices and insight into DNA binding. Structure. 2008; 16(3):468-77. PMC: 2323513. DOI: 10.1016/j.str.2007.12.014. View

12.

Staszewski O, Nikolova T, Kaina B . Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents. Environ Mol Mutagen. 2008; 49(9):734-40. DOI: 10.1002/em.20430. View

13.

Schreiber V, Dantzer F, Ame J, de Murcia G . Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 2006; 7(7):517-28. DOI: 10.1038/nrm1963. View

14.

Mahaney B, Meek K, Lees-Miller S . Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J. 2009; 417(3):639-50. PMC: 2975036. DOI: 10.1042/BJ20080413. View

15.

Downs J, Jackson S . A means to a DNA end: the many roles of Ku. Nat Rev Mol Cell Biol. 2004; 5(5):367-78. DOI: 10.1038/nrm1367. View

16.

Veuger S, Curtin N, Richardson C, Smith G, Durkacz B . Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res. 2003; 63(18):6008-15. View

17.

Meek K, Douglas P, Cui X, Ding Q, Lees-Miller S . trans Autophosphorylation at DNA-dependent protein kinase's two major autophosphorylation site clusters facilitates end processing but not end joining. Mol Cell Biol. 2007; 27(10):3881-90. PMC: 1899996. DOI: 10.1128/MCB.02366-06. View

18.

Perrault R, Wang H, Wang M, Rosidi B, Iliakis G . Backup pathways of NHEJ are suppressed by DNA-PK. J Cell Biochem. 2004; 92(4):781-94. DOI: 10.1002/jcb.20104. View

19.

Ruscetti T, Lehnert B, Halbrook J, Le Trong H, Hoekstra M, Chen D . Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase. J Biol Chem. 1998; 273(23):14461-7. DOI: 10.1074/jbc.273.23.14461. View

20.

Fong P, Boss D, Yap T, Tutt A, Wu P, Mergui-Roelvink M . Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med. 2009; 361(2):123-34. DOI: 10.1056/NEJMoa0900212. View