Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair


Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair1. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality2, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme3, or Escherichia coli LigA, an NAD+-dependent ligase1. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells4,5,6. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.

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Figure 1: Mitochondrial Lig3 activity is critical for cellular viability.
Figure 2: Mitochondrial DNA ligase activity can be provided by a variety of DNA ligases.
Figure 3: Loss of Lig3 is not associated with sensitivity to several DNA-damaging agents or with increased SCE.


  1. 1

    Ellenberger, T. & Tomkinson, A. E. Eukaryotic DNA ligases: structural and functional insights. Annu. Rev. Biochem. 77, 313–338 (2008)

  2. 2

    Puebla-Osorio, N., Lacey, D. B., Alt, F. W. & Zhu, C. Early embryonic lethality due to targeted inactivation of DNA ligase III. Mol. Cell. Biol. 26, 3935–3941 (2006)

  3. 3

    Ho, C. K., Van Etten, J. L. & Shuman, S. Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1. J. Virol. 71, 1931–1937 (1997)

  4. 4

    Thompson, L. H., Brookman, K. W., Jones, N. J., Allen, S. A. & Carrano, A. V. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. Mol. Cell. Biol. 10, 6160–6171 (1990)

  5. 5

    Tebbs, R. S. et al. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev. Biol. 208, 513–529 (1999)

  6. 6

    Lee, Y. et al. The genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1. Nature Neurosci. 12, 973–980 (2009)

  7. 7

    Caldecott, K. W., McKeown, C. K., Tucker, J. D., Ljungquist, S. & Thompson, L. H. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol. 14, 68–76 (1994)

  8. 8

    Mortusewicz, O., Rothbauer, U., Cardoso, M. C. & Leonhardt, H. Differential recruitment of DNA ligase I and III to DNA repair sites. Nucleic Acids Res. 34, 3523–3532 (2006)

  9. 9

    Chen, X. et al. Distinct kinetics of human DNA ligases I, IIIα, IIIβ, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair. DNA Repair (Amst.) 8, 961–968 (2009)

  10. 10

    Leppard, J. B., Dong, Z., Mackey, Z. B. & Tomkinson, A. E. Physical and functional interaction between DNA ligase IIIα and poly(ADP-ribose) polymerase 1 in DNA single-strand break repair. Mol. Cell. Biol. 23, 5919–5927 (2003)

  11. 11

    Lakshmipathy, U. & Campbell, C. The human DNA ligase III gene encodes nuclear and mitochondrial proteins. Mol. Cell. Biol. 19, 3869–3876 (1999)

  12. 12

    Willer, M., Rainey, M., Pullen, T. & Stirling, C. J. The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I. Curr. Biol. 9, 1085–1094 (1999)

  13. 13

    Lakshmipathy, U. & Campbell, C. Mitochondrial DNA ligase III function is independent of Xrcc1. Nucleic Acids Res. 28, 3880–3886 (2000)

  14. 14

    Rossi, M. N. et al. Mitochondrial localization of PARP-1 requires interaction with mitofilin and is involved in the maintenance of mitochondrial DNA integrity. J. Biol. Chem. 284, 31616–31624 (2009)

  15. 15

    Mackey, Z. B. et al. DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly(ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III. J. Biol. Chem. 274, 21679–21687 (1999)

  16. 16

    Cotner-Gohara, E., Kim, I. K., Tomkinson, A. E. & Ellenberger, T. Two DNA-binding and nick recognition modules in human DNA ligase III. J. Biol. Chem. 283, 10764–10772 (2008)

  17. 17

    Santos, J. H., Meyer, J. N., Mandavilli, B. S. & Van Houten, B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 314, 183–199 (2006)

  18. 18

    Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007)

  19. 19

    Park, U. E., Olivera, B. M., Hughes, K. T., Roth, J. R. & Hillyard, D. R. DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium . J. Bacteriol. 171, 2173–2180 (1989)

  20. 20

    Sriskanda, V., Schwer, B., Ho, C. K. & Shuman, S. Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4. Nucleic Acids Res. 27, 3953–3963 (1999)

  21. 21

    Henderson, B. R. & Eleftheriou, A. A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. Exp. Cell Res. 256, 213–224 (2000)

  22. 22

    Moser, J. et al. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase IIIα in a cell-cycle-specific manner. Mol. Cell 27, 311–323 (2007)

  23. 23

    Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005)

  24. 24

    Bentley, D. et al. DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability. Nature Genet. 13, 489–491 (1996)

  25. 25

    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)

  26. 26

    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)

  27. 27

    Gao, Y. et al. DNA ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair. Nature 10.1038/nature09773 (this issue)

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We thank K. Caldecott for the gift of the Lig3 expression vector, and M. Sanz for initial assistance with the SCE analysis. We also thank the members of Jasin laboratory, especially Y. Akamatsu, J. LaRocque, E. Kass and F. Vanoli, for discussions. This work was supported by PA CURE (to B.V.H.) and by National Institutes of Health grants ES019566 (to B.V.H.), NS37956 and CA21765 (to P.J.M.), and GM54668 (to M.J.).

Author information

D.S. performed most of the experiments. D.S. and M.J. designed the research and wrote the paper. A.F. performed the long-range qPCR assays to investigate mitochondrial BER and mitochondrial DNA maintenance, and with B.V.H. analysed the data. Y.G. and P.J.M. designed the initial Lig3 targeting scheme and generated the Lig3wt/cKO embryonic stem cells. J.A. and A.-K.H. acquired confocal images for GFP-tagged proteins. E.B. contributed technical assistance and preparation of the manuscript. S.S. contributed discussions, provided reagents and shared unpublished data.

Correspondence to Maria Jasin.

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Simsek, D., Furda, A., Gao, Y. et al. Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair. Nature 471, 245–248 (2011).

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