Human DNA ligase I completely encircles and partially unwinds nicked DNA

Article metrics


The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5′ adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein–protein interface that may coordinate the joining of Okazaki fragments.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Function and organization of Lig1.
Figure 2: Lig1 intimately engages its DNA substrate.
Figure 3: Lig1 engages the minor groove of DNA.
Figure 4: Ligation fidelity.
Figure 5: Two active conformations of the OBD.


  1. 1

    Lehman, I. R. DNA ligase: structure, mechanism, and function. Science 186, 790–797 (1974)

  2. 2

    Shuman, S. & Schwer, B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol. Microbiol. 17, 405–410 (1995)

  3. 3

    Kodama, K., Barnes, D. E. & Lindahl, T. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I. Nucleic Acids Res. 19, 6093–6099 (1991)

  4. 4

    Luo, J. & Barany, F. Identification of essential residues in Thermus thermophilus DNA ligase. Nucleic Acids Res. 24, 3079–3085 (1996)

  5. 5

    Sriskanda, V. & Shuman, S. Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI. Nucleic Acids Res. 26, 4618–4625 (1998)

  6. 6

    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)

  7. 7

    Sriskanda, V. & Shuman, S. Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase. Nucleic Acids Res. 30, 903–911 (2002)

  8. 8

    Subramanya, H. S., Doherty, A. J., Ashford, S. R. & Wigley, D. B. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell 85, 607–615 (1996)

  9. 9

    Singleton, M. R., Hakansson, K., Timson, D. J. & Wigley, D. B. Structure of the adenylation domain of an NAD+-dependent DNA ligase. Struct. Fold. Des. 7, 35–42 (1999)

  10. 10

    Lee, J. Y. et al. Crystal structure of NAD(+ )-dependent DNA ligase: modular architecture and functional implications. EMBO J. 19, 1119–1129 (2000)

  11. 11

    Odell, M., Sriskanda, V., Shuman, S. & Nikolov, D. B. Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining. Mol. Cell 6, 1183–1193 (2000)

  12. 12

    Doherty, A. J. & Suh, S. W. Structural and mechanistic conservation in DNA ligases. Nucleic Acids Res. 28, 4051–4058 (2000)

  13. 13

    Timson, D. J., Singleton, M. R. & Wigley, D. B. DNA ligases in the repair and replication of DNA. Mutat. Res. 460, 301–318 (2000)

  14. 14

    Martin, I. V. & MacNeill, S. A. ATP-dependent DNA ligases. Genome Biol. 3, Reviews 3005 (2002)

  15. 15

    Tomkinson, A. E., Tappe, N. J. & Friedberg, E. C. DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product. Biochemistry 31, 11762–11771 (1992)

  16. 16

    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)

  17. 17

    Grawunder, U., Zimmer, D. & Leiber, M. R. DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains. Curr. Biol. 8, 873–876 (1998)

  18. 18

    Jeon, H. J. et al. Mutational analyses of the thermostable NAD(+ )-dependent DNA ligase from Thermus filiformis. FEMS Microbiol. Lett. 237, 111–118 (2004)

  19. 19

    Sriskanda, V. & Shuman, S. Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+. J. Biol. Chem. 277, 9695–9700 (2002)

  20. 20

    Gajiwala, K. S. & Pinko, C. Structural rearrangement accompanying NAD(+ ) synthesis within a bacterial DNA ligase crystal. Structure 12, 1449–1459 (2004)

  21. 21

    Hakansson, K., Doherty, A. J., Shuman, S. & Wigley, D. B. X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell 89, 545–553 (1997)

  22. 22

    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)

  23. 23

    Prigent, C., Satoh, M. S., Daly, G., Barnes, D. E. & Lindahl, T. Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I. Mol. Cell. Biol. 14, 310–317 (1994)

  24. 24

    Harrison, C., Ketchen, A. M., Redhead, N. J., O'Sullivan, M. J. & Melton, D. W. Replication failure, genome instability, and increased cancer susceptibility in mice with a point mutation in the DNA ligase I gene. Cancer Res. 62, 4065–4074 (2002)

  25. 25

    Robinson, H. et al. The hyperthermophile chromosomal protein Sac7d sharply kinks DNA. Nature 392, 202–205 (1998)

  26. 26

    Odell, M. & Shuman, S. Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA. J. Biol. Chem. 274, 14032–14039 (1999)

  27. 27

    Doherty, A. J. & Dafforn, T. R. Nick recognition by DNA ligases. J. Mol. Biol. 296, 43–56 (2000)

  28. 28

    Sekiguchi, J. & Shuman, S. Ligation of RNA-containing duplexes by vaccinia DNA ligase. Biochemistry 36, 9073–9079 (1997)

  29. 29

    Rumbaugh, J. A., Murante, R. S., Shi, S. & Bambara, R. A. Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing. J. Biol. Chem. 272, 22591–22599 (1997)

  30. 30

    Sriskanda, V. & Shuman, S. Specificity and fidelity of strand joining by Chlorella virus DNA ligase. Nucleic Acids Res. 26, 3536–3541 (1998)

  31. 31

    Shuman, S. Vaccinia virus DNA ligase: specificity, fidelity, and inhibition. Biochemistry 34, 16138–16147 (1995)

  32. 32

    Bhagwat, A. S., Sanderson, R. J. & Lindahl, T. Delayed DNA joining at 3′ mismatches by human DNA ligases. Nucleic Acids Res. 27, 4028–4033 (1999)

  33. 33

    Liu, P., Burdzy, A. & Sowers, L. C. DNA ligases ensure fidelity by interrogating minor groove contacts. Nucleic Acids Res. 32, 4503–4511 (2004)

  34. 34

    Corbett, K. D. & Berger, J. M. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 33, 95–118 (2004)

  35. 35

    Montecucco, A. & Ciarrocchi, G. AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism. Nucleic Acids Res. 16, 7369–7381 (1988)

  36. 36

    Doherty, A. J. & Wigley, D. B. Functional domains of an ATP-dependent DNA ligase. J. Mol. Biol. 285, 63–71 (1999)

  37. 37

    Warbrick, E. PCNA binding through a conserved motif. Bioessays 20, 195–199 (1998)

  38. 38

    Levin, D. S., McKenna, A. E., Motycka, T. A., Matsumoto, Y. & Tomkinson, A. E. Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair. Curr. Biol. 10, 919–922 (2000)

  39. 39

    Levin, D. S., Bai, W., Yao, N., O'Donnell, M. & Tomkinson, A. E. An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining. Proc. Natl Acad. Sci. USA 94, 12863–12868 (1997)

  40. 40

    Dionne, I., Nookala, R. K., Jackson, S. P., Doherty, A. J. & Bell, S. D. A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus. Mol. Cell 11, 275–282 (2003)

  41. 41

    Teraoka, H. et al. Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct. J. Biol. Chem. 268, 24156–24162 (1993)

  42. 42

    Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993)

  43. 43

    Otwinowski, Z. & Minor, W. in Methods Enzymology (eds Carter, C. W. & Sweet, R. M.) 307–326 (Academic, New York, 1997)

  44. 44

    Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999)

  45. 45

    La Fortelle, E. D. & Bricogne, G. in Methods Enzymology (eds Sweet, R. M. & Carter, C. W.) 472–494 (Academic, New York, 1997)

  46. 46

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  47. 47

    Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998)

  48. 48

    Murshudov, G. N. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997)

  49. 49

    Montecucco, A. et al. The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication. EMBO J. 14, 5379–5386 (1995)

  50. 50

    Dimitriadis, E. K. et al. Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase β. J. Biol. Chem. 273, 20540–20550 (1998)

Download references


X-ray data were measured at beamlines X-12C and X-25 of the National Synchrotron Light Source (Upton, New York), and the SIBYLS beamline 12.3.1 of the Advanced Light Source (Berkeley, California), which are supported by the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and the National Center for Research Resources of the National Institutes of Health. This work was supported by the Structural Cell Biology of DNA Repair Program Grant from the National Cancer Institute, and research grants from the National Institute of General Medical Sciences awarded to T.E. and A.E.T. J.M.P. and P.J.O. are supported by NRSA postdoctoral fellowships from the National Institutes of Health. T.E. is the Hsien Wu and Daisy Yen Wu Professor at Harvard Medical School.

Author information

Correspondence to Tom Ellenberger.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Tables, Figures and Methods

This file contains supplemantary tables 1-3, figures 1–4 and their legends and supplementary methods. (PDF 541 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pascal, J., O'Brien, P., Tomkinson, A. et al. Human DNA ligase I completely encircles and partially unwinds nicked DNA. Nature 432, 473–478 (2004) doi:10.1038/nature03082

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.