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. 1999 Mar 1;18(5):1114–1123. doi: 10.1093/emboj/18.5.1114

Structure of DNA-dependent protein kinase: implications for its regulation by DNA.

K K Leuther 1, O Hammarsten 1, R D Kornberg 1, G Chu 1
PMCID: PMC1171203  PMID: 10064579

Abstract

DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.

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Selected References

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  1. Agard D. A. A least-squares method for determining structure factors in three-dimensional tilted-view reconstructions. J Mol Biol. 1983 Jul 15;167(4):849–852. doi: 10.1016/s0022-2836(83)80114-4. [DOI] [PubMed] [Google Scholar]
  2. Amos L. A., Henderson R., Unwin P. N. Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol. 1982;39(3):183–231. doi: 10.1016/0079-6107(83)90017-2. [DOI] [PubMed] [Google Scholar]
  3. Asturias F. J., Kornberg R. D. A novel method for transfer of two-dimensional crystals from the air/water interface to specimen grids. EM sample preparation/lipid-layer crystallization. J Struct Biol. 1995 Jan-Feb;114(1):60–66. doi: 10.1006/jsbi.1995.1005. [DOI] [PubMed] [Google Scholar]
  4. Baumann P., West S. C. DNA end-joining catalyzed by human cell-free extracts. Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14066–14070. doi: 10.1073/pnas.95.24.14066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bentley N. J., Holtzman D. A., Flaggs G., Keegan K. S., DeMaggio A., Ford J. C., Hoekstra M., Carr A. M. The Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J. 1996 Dec 2;15(23):6641–6651. [PMC free article] [PubMed] [Google Scholar]
  6. Biedermann K. A., Sun J. R., Giaccia A. J., Tosto L. M., Brown J. M. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1394–1397. doi: 10.1073/pnas.88.4.1394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell. 1995 Mar 10;80(5):813–823. doi: 10.1016/0092-8674(95)90360-7. [DOI] [PubMed] [Google Scholar]
  8. Blunt T., Gell D., Fox M., Taccioli G. E., Lehmann A. R., Jackson S. P., Jeggo P. A. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10285–10290. doi: 10.1073/pnas.93.19.10285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bochkarev A., Pfuetzner R. A., Edwards A. M., Frappier L. Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature. 1997 Jan 9;385(6612):176–181. doi: 10.1038/385176a0. [DOI] [PubMed] [Google Scholar]
  10. Bosma M. J., Carroll A. M. The SCID mouse mutant: definition, characterization, and potential uses. Annu Rev Immunol. 1991;9:323–350. doi: 10.1146/annurev.iy.09.040191.001543. [DOI] [PubMed] [Google Scholar]
  11. Boubnov N. V., Weaver D. T. scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase. Mol Cell Biol. 1995 Oct;15(10):5700–5706. doi: 10.1128/mcb.15.10.5700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brisson A., Olofsson A., Ringler P., Schmutz M., Stoylova S. Two-dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography. Biol Cell. 1994;80(2-3):221–228. [PubMed] [Google Scholar]
  13. Ceska T. A., Sayers J. R., Stier G., Suck D. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease. Nature. 1996 Jul 4;382(6586):90–93. doi: 10.1038/382090a0. [DOI] [PubMed] [Google Scholar]
  14. Chan D. W., Lees-Miller S. P. The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J Biol Chem. 1996 Apr 12;271(15):8936–8941. doi: 10.1074/jbc.271.15.8936. [DOI] [PubMed] [Google Scholar]
  15. Chu G. Double strand break repair. J Biol Chem. 1997 Sep 26;272(39):24097–24100. doi: 10.1074/jbc.272.39.24097. [DOI] [PubMed] [Google Scholar]
  16. Cimprich K. A., Shin T. B., Keith C. T., Schreiber S. L. cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2850–2855. doi: 10.1073/pnas.93.7.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Critchlow S. E., Bowater R. P., Jackson S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol. 1997 Aug 1;7(8):588–598. doi: 10.1016/s0960-9822(06)00258-2. [DOI] [PubMed] [Google Scholar]
  18. Danska J. S., Holland D. P., Mariathasan S., Williams K. M., Guidos C. J. Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol Cell Biol. 1996 Oct;16(10):5507–5517. doi: 10.1128/mcb.16.10.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Darst S. A., Edwards A. M., Kubalek E. W., Kornberg R. D. Three-dimensional structure of yeast RNA polymerase II at 16 A resolution. Cell. 1991 Jul 12;66(1):121–128. doi: 10.1016/0092-8674(91)90144-n. [DOI] [PubMed] [Google Scholar]
  20. Falzon M., Fewell J. W., Kuff E. L. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J Biol Chem. 1993 May 15;268(14):10546–10552. [PubMed] [Google Scholar]
  21. Fried L. M., Koumenis C., Peterson S. R., Green S. L., van Zijl P., Allalunis-Turner J., Chen D. J., Fishel R., Giaccia A. J., Brown J. M. The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: replication protein A hyperphosphorylation and p53 induction. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13825–13830. doi: 10.1073/pnas.93.24.13825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fulop G. M., Phillips R. A. The scid mutation in mice causes a general defect in DNA repair. Nature. 1990 Oct 4;347(6292):479–482. doi: 10.1038/347479a0. [DOI] [PubMed] [Google Scholar]
  23. Gao Y., Chaudhuri J., Zhu C., Davidson L., Weaver D. T., Alt F. W. A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity. 1998 Sep;9(3):367–376. doi: 10.1016/s1074-7613(00)80619-6. [DOI] [PubMed] [Google Scholar]
  24. Giffin W., Kwast-Welfeld J., Rodda D. J., Préfontaine G. G., Traykova-Andonova M., Zhang Y., Weigel N. L., Lefebvre Y. A., Haché R. J. Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor. J Biol Chem. 1997 Feb 28;272(9):5647–5658. doi: 10.1074/jbc.272.9.5647. [DOI] [PubMed] [Google Scholar]
  25. Glaeser R. M., Downing K. H. Assessment of resolution in biological electron crystallography. Ultramicroscopy. 1992 Nov;47(1-3):256–265. doi: 10.1016/0304-3991(92)90201-t. [DOI] [PubMed] [Google Scholar]
  26. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131–142. doi: 10.1016/0092-8674(93)90057-w. [DOI] [PubMed] [Google Scholar]
  27. Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature. 1997 Jul 31;388(6641):492–495. doi: 10.1038/41358. [DOI] [PubMed] [Google Scholar]
  28. Hammarsten O., Chu G. DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):525–530. doi: 10.1073/pnas.95.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  30. Hessler D., Young S. J., Carragher B. O., Martone M. E., Lamont S., Whittaker M., Milligan R. A., Masliah E., Hinshaw J. E., Ellisman M. H. Programs for visualization in three-dimensional microscopy. Neuroimage. 1992 Aug;1(1):55–67. doi: 10.1016/1053-8119(92)90007-a. [DOI] [PubMed] [Google Scholar]
  31. Jap B. K., Zulauf M., Scheybani T., Hefti A., Baumeister W., Aebi U., Engel A. 2D crystallization: from art to science. Ultramicroscopy. 1992 Oct;46(1-4):45–84. doi: 10.1016/0304-3991(92)90007-7. [DOI] [PubMed] [Google Scholar]
  32. Jensen G. J., Meredith G., Bushnell D. A., Kornberg R. D. Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7. EMBO J. 1998 Apr 15;17(8):2353–2358. doi: 10.1093/emboj/17.8.2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Joyce C. M., Steitz T. A. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. doi: 10.1146/annurev.bi.63.070194.004021. [DOI] [PubMed] [Google Scholar]
  34. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. doi: 10.1126/science.7855601. [DOI] [PubMed] [Google Scholar]
  35. Korolev S., Hsieh J., Gauss G. H., Lohman T. M., Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell. 1997 Aug 22;90(4):635–647. doi: 10.1016/s0092-8674(00)80525-5. [DOI] [PubMed] [Google Scholar]
  36. Kovall R., Matthews B. W. Toroidal structure of lambda-exonuclease. Science. 1997 Sep 19;277(5333):1824–1827. doi: 10.1126/science.277.5333.1824. [DOI] [PubMed] [Google Scholar]
  37. Leber R., Wise T. W., Mizuta R., Meek K. The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. J Biol Chem. 1998 Jan 16;273(3):1794–1801. doi: 10.1074/jbc.273.3.1794. [DOI] [PubMed] [Google Scholar]
  38. Leuther K. K., Bushnell D. A., Kornberg R. D. Two-dimensional crystallography of TFIIB- and IIE-RNA polymerase II complexes: implications for start site selection and initiation complex formation. Cell. 1996 May 31;85(5):773–779. doi: 10.1016/s0092-8674(00)81242-8. [DOI] [PubMed] [Google Scholar]
  39. Lieber M. R., Hesse J. E., Lewis S., Bosma G. C., Rosenberg N., Mizuuchi K., Bosma M. J., Gellert M. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell. 1988 Oct 7;55(1):7–16. doi: 10.1016/0092-8674(88)90004-9. [DOI] [PubMed] [Google Scholar]
  40. Mimori T., Hardin J. A. Mechanism of interaction between Ku protein and DNA. J Biol Chem. 1986 Aug 5;261(22):10375–10379. [PubMed] [Google Scholar]
  41. Peterson S. R., Kurimasa A., Oshimura M., Dynan W. S., Bradbury E. M., Chen D. J. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3171–3174. doi: 10.1073/pnas.92.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rathmell W. K., Chu G. A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol Cell Biol. 1994 Jul;14(7):4741–4748. doi: 10.1128/mcb.14.7.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rathmell W. K., Chu G. Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7623–7627. doi: 10.1073/pnas.91.16.7623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Savitsky K., Bar-Shira A., Gilad S., Rotman G., Ziv Y., Vanagaite L., Tagle D. A., Smith S., Uziel T., Sfez S. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995 Jun 23;268(5218):1749–1753. doi: 10.1126/science.7792600. [DOI] [PubMed] [Google Scholar]
  45. Smider V., Rathmell W. K., Brown G., Lewis S., Chu G. Failure of hairpin-ended and nicked DNA To activate DNA-dependent protein kinase: implications for V(D)J recombination. Mol Cell Biol. 1998 Nov;18(11):6853–6858. doi: 10.1128/mcb.18.11.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smider V., Rathmell W. K., Lieber M. R., Chu G. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science. 1994 Oct 14;266(5183):288–291. doi: 10.1126/science.7939667. [DOI] [PubMed] [Google Scholar]
  47. Sousa R. Structural and mechanistic relationships between nucleic acid polymerases. Trends Biochem Sci. 1996 May;21(5):186–190. [PubMed] [Google Scholar]
  48. Taccioli G. E., Amatucci A. G., Beamish H. J., Gell D., Xiang X. H., Torres Arzayus M. I., Priestley A., Jackson S. P., Marshak Rothstein A., Jeggo P. A. Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity. 1998 Sep;9(3):355–366. doi: 10.1016/s1074-7613(00)80618-4. [DOI] [PubMed] [Google Scholar]
  49. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  50. Tuteja N., Tuteja R., Ochem A., Taneja P., Huang N. W., Simoncsits A., Susic S., Rahman K., Marusic L., Chen J. Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J. 1994 Oct 17;13(20):4991–5001. doi: 10.1002/j.1460-2075.1994.tb06826.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Valpuesta J. M., Carrascosa J. L., Henderson R. Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. J Mol Biol. 1994 Jul 22;240(4):281–287. doi: 10.1006/jmbi.1994.1445. [DOI] [PubMed] [Google Scholar]
  52. Yaneva M., Kowalewski T., Lieber M. R. Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies. EMBO J. 1997 Aug 15;16(16):5098–5112. doi: 10.1093/emboj/16.16.5098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yavuzer U., Smith G. C., Bliss T., Werner D., Jackson S. P. DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D. Genes Dev. 1998 Jul 15;12(14):2188–2199. doi: 10.1101/gad.12.14.2188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zakian V. A. ATM-related genes: what do they tell us about functions of the human gene? Cell. 1995 Sep 8;82(5):685–687. doi: 10.1016/0092-8674(95)90463-8. [DOI] [PubMed] [Google Scholar]

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