Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Jan 3;92(1):320–324. doi: 10.1073/pnas.92.1.320

DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair.

N J Finnie 1, T M Gottlieb 1, T Blunt 1, P A Jeggo 1, S P Jackson 1
PMCID: PMC42870  PMID: 7816841

Abstract

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit called p350 and a DNA binding component termed Ku. Ku consists of two tightly associated polypeptides of approximately 70 kDa and 80 kDa (Ku80). An intriguing feature of DNA-PK is that it binds to DNA ends and other discontinuities in DNA and requires these structures for its activation. This suggests that DNA-PK may function in DNA repair and/or recombination. Consistent with this, Ku DNA binding activity was shown recently to be absent in extracts of hamster xrs-6 cells, which are defective in DNA double-strand (ds) break repair and V(D)J recombination. Furthermore, xrs-6 cells are complemented by expression of the Ku80 cDNA. To date, DNA-PK activity has been demonstrated unequivocally only in extracts of primate cells. Here, we describe an assay that can detect DNA-PK activity in extracts of mouse, hamster, Xenopus, and Drosophila cells. Using this assay, we find that xrs-6 cells completely lack DNA-PK activity. By contrast, xrs-6 derivatives complemented by human chromosome fragments bearing the Ku80 gene have restored both the DNA end binding and kinase activities associated with DNA-PK. Finally, we show that xrs-6 extracts are complemented biochemically by purified Ku. Our findings indicate that the xrs-6 defects are direct consequences of the mutation in Ku80 and implicate DNA-PK in recombination and DNA repair processes.

Full text

PDF
320

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson C. W. DNA damage and the DNA-activated protein kinase. Trends Biochem Sci. 1993 Nov;18(11):433–437. doi: 10.1016/0968-0004(93)90144-c. [DOI] [PubMed] [Google Scholar]
  2. Anderson C. W., Lees-Miller S. P. The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr. 1992;2(4):283–314. [PubMed] [Google Scholar]
  3. Bannister A. J., Gottlieb T. M., Kouzarides T., Jackson S. P. c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif. Nucleic Acids Res. 1993 Mar 11;21(5):1289–1295. doi: 10.1093/nar/21.5.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cai Q. Q., Plet A., Imbert J., Lafage-Pochitaloff M., Cerdan C., Blanchard J. M. Chromosomal location and expression of the genes coding for Ku p70 and p80 in human cell lines and normal tissues. Cytogenet Cell Genet. 1994;65(4):221–227. doi: 10.1159/000133635. [DOI] [PubMed] [Google Scholar]
  5. Carter T., Vancurová I., Sun I., Lou W., DeLeon S. A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol. 1990 Dec;10(12):6460–6471. doi: 10.1128/mcb.10.12.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DePamphili M. L. How transcription factors regulate origins of DNA replication in eukaryotic cells. Trends Cell Biol. 1993 May;3(5):161–167. doi: 10.1016/0962-8924(93)90137-p. [DOI] [PubMed] [Google Scholar]
  7. Drapkin R., Sancar A., Reinberg D. Where transcription meets repair. Cell. 1994 Apr 8;77(1):9–12. doi: 10.1016/0092-8674(94)90228-3. [DOI] [PubMed] [Google Scholar]
  8. Dröge P. Transcription-driven site-specific DNA recombination in vitro. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2759–2763. doi: 10.1073/pnas.90.7.2759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dvir A., Stein L. Y., Calore B. L., Dynan W. S. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J Biol Chem. 1993 May 15;268(14):10440–10447. [PubMed] [Google Scholar]
  10. Falzon M., Kuff E. L. The nucleotide sequence of a mouse cDNA encoding the 80 kDa subunit of the Ku (p70/p80) autoantigen. Nucleic Acids Res. 1992 Jul 25;20(14):3784–3784. doi: 10.1093/nar/20.14.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feldmann H., Winnacker E. L. A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 15;268(17):12895–12900. [PubMed] [Google Scholar]
  12. Finnie N., Gottlieb T., Hartley K., Jackson S. P. Transcription factor phosphorylation by the DNA-dependent protein kinase. Biochem Soc Trans. 1993 Nov;21(4):930–935. doi: 10.1042/bst0210930. [DOI] [PubMed] [Google Scholar]
  13. Getts R. C., Stamato T. D. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem. 1994 Jun 10;269(23):15981–15984. [PubMed] [Google Scholar]
  14. Gottlieb T. M., Jackson S. P. Protein kinases and DNA damage. Trends Biochem Sci. 1994 Nov;19(11):500–503. doi: 10.1016/0968-0004(94)90138-4. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hafezparast M., Kaur G. P., Zdzienicka M., Athwal R. S., Lehmann A. R., Jeggo P. A. Subchromosomal localization of a gene (XRCC5) involved in double strand break repair to the region 2q34-36. Somat Cell Mol Genet. 1993 Sep;19(5):413–421. doi: 10.1007/BF01233246. [DOI] [PubMed] [Google Scholar]
  17. Jackson S. P., MacDonald J. J., Lees-Miller S., Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 1990 Oct 5;63(1):155–165. doi: 10.1016/0092-8674(90)90296-q. [DOI] [PubMed] [Google Scholar]
  18. Jackson S. P. Regulating transcription factor activity by phosphorylation. Trends Cell Biol. 1992 Apr;2(4):104–108. doi: 10.1016/0962-8924(92)90014-e. [DOI] [PubMed] [Google Scholar]
  19. Jacoby D. B., Wensink P. C. Yolk protein factor 1 is a Drosophila homolog of Ku, the DNA-binding subunit of a DNA-dependent protein kinase from humans. J Biol Chem. 1994 Apr 15;269(15):11484–11491. [PubMed] [Google Scholar]
  20. Jeggo P. A., Hafezparast M., Thompson A. F., Broughton B. C., Kaur G. P., Zdzienicka M. Z., Athwal R. S. Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6423–6427. doi: 10.1073/pnas.89.14.6423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jeggo P. A., Kemp L. M. X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents. Mutat Res. 1983 Dec;112(6):313–327. doi: 10.1016/0167-8817(83)90026-3. [DOI] [PubMed] [Google Scholar]
  22. Jeggo P. A., Tesmer J., Chen D. J. Genetic analysis of ionising radiation sensitive mutants of cultured mammalian cell lines. Mutat Res. 1991 Mar;254(2):125–133. doi: 10.1016/0921-8777(91)90003-8. [DOI] [PubMed] [Google Scholar]
  23. Kemp L. M., Sedgwick S. G., Jeggo P. A. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining. Mutat Res. 1984 Nov-Dec;132(5-6):189–196. doi: 10.1016/0167-8817(84)90037-3. [DOI] [PubMed] [Google Scholar]
  24. Lees-Miller S. P., Chen Y. R., Anderson C. W. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol. 1990 Dec;10(12):6472–6481. doi: 10.1128/mcb.10.12.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lees-Miller S. P., Sakaguchi K., Ullrich S. J., Appella E., Anderson C. W. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992 Nov;12(11):5041–5049. doi: 10.1128/mcb.12.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leung H., Maizels N. Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4154–4158. doi: 10.1073/pnas.89.9.4154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller L., Daniel J. C. Comparison of in vivo and in vitro ribosomal RNA synthesis in nucleolar mutants of Xenopus laevis. In Vitro. 1977 Sep;13(9):557–563. doi: 10.1007/BF02627851. [DOI] [PubMed] [Google Scholar]
  28. Morozov V. E., Falzon M., Anderson C. W., Kuff E. L. DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. J Biol Chem. 1994 Jun 17;269(24):16684–16688. [PubMed] [Google Scholar]
  29. Porges A. J., Ng T., Reeves W. H. Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species. J Immunol. 1990 Dec 15;145(12):4222–4228. [PubMed] [Google Scholar]
  30. 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]
  31. Schöler H. R., Hatzopoulos A. K., Balling R., Suzuki N., Gruss P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 1989 Sep;8(9):2543–2550. doi: 10.1002/j.1460-2075.1989.tb08392.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taccioli G. E., Cheng H. L., Varghese A. J., Whitmore G., Alt F. W. A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation. J Biol Chem. 1994 Mar 11;269(10):7439–7442. [PubMed] [Google Scholar]
  33. 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]
  34. Taccioli G. E., Rathbun G., Oltz E., Stamato T., Jeggo P. A., Alt F. W. Impairment of V(D)J recombination in double-strand break repair mutants. Science. 1993 Apr 9;260(5105):207–210. doi: 10.1126/science.8469973. [DOI] [PubMed] [Google Scholar]
  35. Thacker J., Wilkinson R. E. The genetic basis of resistance to ionising radiation damage in cultured mammalian cells. Mutat Res. 1991 Mar;254(2):135–142. doi: 10.1016/0921-8777(91)90004-9. [DOI] [PubMed] [Google Scholar]
  36. Wang Y., Eckhart W. Phosphorylation sites in the amino-terminal region of mouse p53. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4231–4235. doi: 10.1073/pnas.89.10.4231. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES