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Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends

Abstract

Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites)2. At single-strand breaks, 5′-terminal abasic sites are excised by the 5′-deoxyribose-5-phosphate (5′-dRP) lyase activity of DNA polymerase β (pol β)3,4,5,6: here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5′-dRP/AP lyase activity. Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping to distinguish this pathway from otherwise robust alternative NHEJ pathways. The NHEJ factor Ku can be identified as an effective 5′-dRP/AP lyase. In a similar manner to other lyases7, Ku nicks DNA 3′ of an abasic site by a mechanism involving a Schiff-base covalent intermediate with the abasic site. We show by using cell extracts that Ku is essential for the efficient removal of AP sites near double-strand breaks and, consistent with this result, that joining of such breaks is specifically decreased in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.

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Figure 1: NHEJ of ends with abasic sites.
Figure 2: 5′-dRP/AP lyase activity of purified NHEJ factors.
Figure 3: AP lyase activity of cell extracts with or without Ku.

References

  1. 1

    Jeggo, P. A. Studies on mammalian mutants defective in rejoining double-strand breaks in DNA. Mutat. Res. 239, 1–16 (1990)

    CAS  Article  Google Scholar 

  2. 2

    Ward, J. F. The complexity of DNA damage: relevance to biological consequences. Int. J. Radiat. Biol. 66, 427–432 (1994)

    CAS  Article  Google Scholar 

  3. 3

    Matsumoto, Y. & Kim, K. Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science 269, 699–702 (1995)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Prasad, R., Beard, W. A., Strauss, P. R. & Wilson, S. H. Human DNA polymerase β deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism. J. Biol. Chem. 273, 15263–15270 (1998)

    CAS  Article  Google Scholar 

  5. 5

    Sobol, R. W. et al. The lyase activity of the DNA repair protein β-polymerase protects from DNA-damage-induced cytotoxicity. Nature 405, 807–810 (2000)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Allinson, S. L., Dianova, I. I. & Dianov, G. L. DNA polymerase β is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts. EMBO J. 20, 6919–6926 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Piersen, C. E., Prasad, R., Wilson, S. H. & Lloyd, R. S. Evidence for an imino intermediate in the DNA polymerase β deoxyribose phosphate excision reaction. J. Biol. Chem. 271, 17811–17815 (1996)

    CAS  Article  Google Scholar 

  8. 8

    Pogozelski, W. K. & Tullius, T. D. Oxidative strand scission of nucleic acids: routes initiated by hydrogen abstraction from the sugar moiety. Chem. Rev. 98, 1089–1108 (1998)

    CAS  Article  Google Scholar 

  9. 9

    Ahnstrom, G. & Bryant, P. E. DNA double-strand breaks generated by the repair of X-ray damage in Chinese hamster cells. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 41, 671–676 (1982)

    CAS  Article  Google Scholar 

  10. 10

    Yang, N., Galick, H. & Wallace, S. S. Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double-strand breaks. DNA Repair (Amst.) 3, 1323–1334 (2004)

    CAS  Article  Google Scholar 

  11. 11

    Di Noia, J. M. et al. Dependence of antibody gene diversification on uracil excision. J. Exp. Med. 204, 3209–3219 (2007)

    CAS  Article  Google Scholar 

  12. 12

    Rada, C., Di Noia, J. M. & Neuberger, M. S. Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation. Mol. Cell 16, 163–171 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Li, H. et al. Deleting Ku70 is milder than deleting Ku80 in p53-mutant mice and cells. Oncogene 28, 1875–1878 (2009)

    CAS  Article  Google Scholar 

  14. 14

    Kabotyanski, E. B., Gomelsky, L., Han, J. O., Stamato, T. D. & Roth, D. B. Double-strand break repair in Ku86- and XRCC4-deficient cells. Nucleic Acids Res. 26, 5333–5342 (1998)

    CAS  Article  Google Scholar 

  15. 15

    Guirouilh-Barbat, J., Rass, E., Plo, I., Bertrand, P. & Lopez, B. S. Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends. Proc. Natl Acad. Sci. USA 104, 20902–20907 (2007)

    ADS  CAS  Article  Google Scholar 

  16. 16

    McVey, M. & Lee, S. E. MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet. 24, 529–538 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Walker, J. R., Corpina, R. A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Bebenek, K., Garcia-Diaz, M., Patishall, S. R. & Kunkel, T. A. Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV. J. Biol. Chem. 280, 20051–20058 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Prasad, R. et al. Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase β as revealed by site-directed mutagenesis. DNA binding and 5′-deoxyribose phosphate lyase activities. J. Biol. Chem. 273, 11121–11126 (1998)

    CAS  Article  Google Scholar 

  20. 20

    Stewart, R. D. Two-lesion kinetic model of double-strand break rejoining and cell killing. Radiat. Res. 156, 365–378 (2001)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Longley, M. J., Prasad, R., Srivastava, D. K., Wilson, S. H. & Copeland, W. C. Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase gamma and its role in mitochondrial base excision repair in vitro. Proc. Natl Acad. Sci. USA 95, 12244–12248 (1998)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Prasad, R. et al. Human DNA polymerase θ possesses 5′-dRP lyase activity and functions in single-nucleotide base excision repair in vitro. Nucleic Acids Res. 37, 1868–1877 (2009)

    CAS  Article  Google Scholar 

  23. 23

    Ilina, E. S., Lavrik, O. I. & Khodyreva, S. N. Ku antigen interacts with abasic sites. Biochim. Biophys. Acta 1784, 1777–1785 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Garcia-Diaz, M., Bebenek, K., Kunkel, T. A. & Blanco, L. Identification of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in human DNA polymerase λ: a possible role in base excision repair. J. Biol. Chem. 276, 34659–34663 (2001)

    CAS  Article  Google Scholar 

  25. 25

    Daley, J. M. & Wilson, T. E. Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining. DNA Repair (Amst.) 7, 67–76 (2008)

    CAS  Article  Google Scholar 

  26. 26

    Anderson, C. W. & Lees-Miller, S. P. The nuclear serine/threonine protein kinase DNA-PK. Crit. Rev. Eukaryot. Gene Expr. 2, 283–314 (1992)

    CAS  PubMed  Google Scholar 

  27. 27

    Schulte-Uentrop, L., El-Awady, R. A., Schliecker, L., Willers, H. & Dahm-Daphi, J. Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks. Nucleic Acids Res. 36, 2561–2569 (2008)

    CAS  Article  Google Scholar 

  28. 28

    Nick McElhinny, S. A. et al. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol. Cell 19, 357–366 (2005)

    CAS  Article  Google Scholar 

  29. 29

    Ding, Q. et al. Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair. Mol. Cell. Biol. 23, 5836–5848 (2003)

    CAS  Article  Google Scholar 

  30. 30

    Davis, B. J., Havener, J. M. & Ramsden, D. A. End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining. Nucleic Acids Res. 36, 3085–3094 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Hirt, B. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369 (1967)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. Gellert, T. Kunkel, M. Garcia-Diaz, T. Traut, L. Harrison and K. Meek for helpful comments. This work was supported by Public Health Service (PHS) grant CA 84442 and a Leukemia and Lymphoma Society scholar award to D.A.R., and by PHS grants R01 CA76317-05A1 and P01 AG17242 to P.H.

Author Contributions Experiments were designed by S.A.R. and D.A.R. In vitro experiments were performed by S.A.R, N.S., M.D.B. and D.A.R. Mutagenesis and protein purification were performed by S.A.R. and D.A.R. S.A.R., C.S., J.M.H. and M.D.B. performed cellular experiments. P.H. provided Ku70 knockout dermal fibroblasts. S.A.R. and D.A.R. wrote the manuscript with the aid of N.S. and M.D.B.

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Correspondence to Dale A. Ramsden.

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Roberts, S., Strande, N., Burkhalter, M. et al. Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends. Nature 464, 1214–1217 (2010). https://doi.org/10.1038/nature08926

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