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Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C

Nature Structural & Molecular Biology volume 12, pages 304–312 (2005)Cite this article

Abstract

DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5′-overhang DSBs is highly mutagenic (∼50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5′ overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.

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Figure 1: Multiple DNA ligases of Mycobacterium smegmatis.
Figure 2: NHEJ of blunt-end DSBs in M. smegmatis depends on LigD and is error-prone.
Figure 3: Molecular outcome of imprecise NHEJ events at blunt DSBs.
Figure 4: NHEJ of 5′ cohesive ends is imprecise and depends on LigD.
Figure 5: Outcome of imprecise NHEJ events at 5′-overhang DSBs.
Figure 6: Ligase and polymerase activities of recombinant mycobacterium LigD.
Figure 7: Mycobacterial NHEJ requires Ku.

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References

  1. Buchmeier, N.A. et al. DNA repair is more important than catalase for Salmonella virulence in mice. J. Clin. Invest. 95, 1047–1053 (1995).

    Article  CAS  Google Scholar 

  2. Buchmeier, N.A., Lipps, C.J., So, M.Y. & Heffron, F. Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol. Microbiol. 7, 933–936 (1993).

    Article  CAS  Google Scholar 

  3. O'Rourke, E.J. et al. Pathogen DNA as target for host-generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization. Proc. Natl. Acad. Sci. USA 100, 2789–2794 (2003).

    Article  CAS  Google Scholar 

  4. Suvarnapunya, A.E., Lagasse, H.A. & Stein, M.A. The role of DNA base excision repair in the pathogenesis of Salmonella enterica serovar Typhimurium. Mol. Microbiol. 48, 549–559 (2003).

    Article  CAS  Google Scholar 

  5. Sander, P. et al. Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model. Infect. Immun. 69, 3562–3568 (2001).

    Article  CAS  Google Scholar 

  6. Darwin, K.H., Ehrt, S., Gutierrez-Ramos, J.C., Weich, N. & Nathan, C.F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 (2003).

    Article  CAS  Google Scholar 

  7. Boshoff, H.I., Reed, M.B., Barry, C.E. & Mizrahi, V. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183–193 (2003).

    Article  CAS  Google Scholar 

  8. Cromie, G.A., Connelly, J.C. & Leach, D.R. Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. Mol. Cell 8, 1163–1174 (2001).

    Article  CAS  Google Scholar 

  9. Lieber, M.R., Ma, Y., Pannicke, U. & Schwarz, K. Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell Biol. 4, 712–720 (2003).

    Article  CAS  Google Scholar 

  10. Downs, J.A. & Jackson, S.P. A means to a DNA end: the many roles of Ku. Nat. Rev. Mol. Cell Biol. 5, 367–378 (2004).

    Article  CAS  Google Scholar 

  11. Riballo, E. et al. Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient. Curr. Biol. 9, 699–702 (1999).

    Article  CAS  Google Scholar 

  12. Barnes, D.E., Tomkinson, A.E., Lehmann, A.R., Webster, A.D. & Lindahl, T. Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. Cell 69, 495–503 (1992).

    Article  CAS  Google Scholar 

  13. Schar, P., Herrmann, G., Daly, G. & Lindahl, T. A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks. Genes Dev. 11, 1912–1924 (1997).

    Article  CAS  Google Scholar 

  14. Teo, S.H. & Jackson, S.P. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J. 16, 4788–4795 (1997).

    Article  CAS  Google Scholar 

  15. Wilson, T.E., Grawunder, U. & Lieber, M.R. Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature 388, 495–498 (1997).

    Article  CAS  Google Scholar 

  16. Frank, K.M. et al. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177 (1998).

    Article  CAS  Google Scholar 

  17. Grawunder, U., Zimmer, D., Fugmann, S., Schwarz, K. & Lieber, M.R. DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol. Cell 2, 477–484 (1998).

    Article  CAS  Google Scholar 

  18. Konrad, E.B., Modrich, P. & Lehman, I.R. Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase. J. Mol. Biol. 77, 519–529 (1973).

    Article  CAS  Google Scholar 

  19. Magnet, S. & Blanchard, J.S. Mechanistic and kinetic study of the ATP-dependent DNA ligase of Neisseria meningitidis. Biochemistry 43, 710–717 (2004).

    Article  CAS  Google Scholar 

  20. Wilkinson, A., Day, J. & Bowater, R. Bacterial DNA ligases. Mol. Microbiol. 40, 1241–1248 (2001).

    Article  CAS  Google Scholar 

  21. Cheng, C. & Shuman, S. Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae. Nucleic Acids Res. 25, 1369–1374 (1997).

    Article  CAS  Google Scholar 

  22. Gong, C., Martins, A., Bongiorno, P., Glickman, M. & Shuman, S. Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J. Biol. Chem. 279, 20594–20606 (2004).

    Article  CAS  Google Scholar 

  23. Aravind, L. & Koonin, E.V. Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res. 11, 1365–1374 (2001).

    Article  CAS  Google Scholar 

  24. Doherty, A.J., Jackson, S.P. & Weller, G.R. Identification of bacterial homologues of the Ku DNA repair proteins. FEBS Lett. 500, 186–188 (2001).

    Article  CAS  Google Scholar 

  25. Della, M. et al. Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. Science 306, 683–685 (2004).

    Article  CAS  Google Scholar 

  26. Zhu, H. & Shuman, S. A primer-dependent polymerase function of Pseudomonas aeruginosa ATP-dependent DNA ligase (LigD). J. Biol. Chem. 280, 418–427 (2005).

    Article  CAS  Google Scholar 

  27. Weller, G.R. et al. Identification of a DNA nonhomologous end-joining complex in bacteria. Science 297, 1686–1689 (2002).

    Article  CAS  Google Scholar 

  28. Braunstein, M., Brown, A.M., Kurtz, S. & Jacobs, W.R. Jr. Two nonredundant SecA homologues function in mycobacteria. J. Bacteriol. 183, 6979–6990 (2001).

    Article  CAS  Google Scholar 

  29. Lipps, G., Weinzierl, A.O., von Scheven, G., Buchen, C. & Cramer, P. Structure of a bifunctional DNA primase-polymerase. Nat. Struct. Mol. Biol. 11, 157–162 (2004).

    Article  CAS  Google Scholar 

  30. Ito, N., Nureki, O., Shirouzu, M., Yokoyama, S. & Hanaoka, F. Crystal structure of the Pyrococcus horikoshii DNA primase-UTP complex: implications for the mechanism of primer synthesis. Genes Cells 8, 913–923 (2003).

    Article  CAS  Google Scholar 

  31. Augustin, M.A., Huber, R. & Kaiser, J.T. Crystal structure of a DNA-dependent RNA polymerase (DNA primase). Nat. Struct. Biol. 8, 57–61 (2001).

    Article  CAS  Google Scholar 

  32. Glickman, M.S. The mmaA2 gene of Mycobacterium tuberculosis encodes the distal cyclopropane synthase of the α-mycolic acid. J. Biol. Chem. 278, 7844–7849 (2003).

    Article  CAS  Google Scholar 

  33. Manolis, K.G. et al. Novel functional requirements for non-homologous DNA end joining in Schizosaccharomyces pombe. EMBO J. 20, 210–221 (2001).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Aylon, Y., Liefshitz, B. & Kupiec, M. The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle. EMBO J. 23, 4868–4875 (2004).

    Article  CAS  Google Scholar 

  36. Heidenreich, E., Novotny, R., Kneidinger, B., Holzmann, V. & Wintersberger, U. Non-homologous end joining as an important mutagenic process in cell cycle– arrested cells. EMBO J. 22, 2274–2283 (2003).

    Article  CAS  Google Scholar 

  37. Ferreira, M.G. & Cooper, J.P. Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle. Genes Dev. 18, 2249–2254 (2004).

    Article  CAS  Google Scholar 

  38. Timm, J., Lim, E.M. & Gicquel, B. Escherichia coli– mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. J. Bacteriol. 176, 6749–6753 (1994).

    Article  CAS  Google Scholar 

  39. Golemis, E. et al. The interaction trap. In Current Protocols in Molecular Biology (eds. Ausubel, F.M. et al.) 20.1.1–20.1.2 (Wiley, New York, 1999).

    Google Scholar 

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Acknowledgements

This research was supported by US National Institutes of Health grants AI53417 (to M.S.G.) and GM63611 (to S.S.). M.S.G. is the recipient of research awards from the Ellison Medical Foundation and the New York Academy of Medicine Speakers Fund for Biomedical Research. S.S. is an American Cancer Society Research Professor.

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Author notes

  1. Chunling Gong and Paola Bongiorno: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunology and Molecular Biology Programs, Sloan-Kettering Institute, New York, 10021, New York, USA

    Chunling Gong, Paola Bongiorno, Alexandra Martins, Nicolas C Stephanou, Hui Zhu, Stewart Shuman & Michael S Glickman

  2. Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, New York, 10021, New York, USA

    Chunling Gong, Paola Bongiorno, Alexandra Martins, Nicolas C Stephanou, Hui Zhu, Stewart Shuman & Michael S Glickman

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Correspondence to Stewart Shuman or Michael S Glickman.

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Supplementary information

Supplementary Table 1

Mycobacterium smegmatis deletion strains. (PDF 19 kb)

Supplementary Methods (PDF 89 kb)

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Gong, C., Bongiorno, P., Martins, A. et al. Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat Struct Mol Biol 12, 304–312 (2005). https://doi.org/10.1038/nsmb915

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