Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I

Abstract

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 Å resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.

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Figure 1: Amino acid sequence of T7 endonuclease I expressed in E. coli and stereo views showing representative portions of the final refined, 2Fo − Fc electron density maps at 2.1 Å.
Figure 2: Structure and active site of endonuclease I.
Figure 3: Comparison of the active site of endonuclease I with restriction endonucleases.
Figure 4: Comparison of the structures of a, T7 endonuclease I, b, T4 endonuclease VII (PDB entry 1EN7) and c, RuvC (PDB entry 1HJR) viewed along their two-fold symmetry axes.

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References

  1. 1

    Holliday, R. Genet. Res, 5, 282–304 (1964).

  2. 2

    Orr-Weaver, T.L., Szostak, J.W. & Rothstein, R.J. Proc. Natl. Acad. Sci. USA 78, 6354–6358 (1981).

  3. 3

    Potter, H. & Dressler, D. Proc. Natl. Acad. Sci. USA 73, 3000–3004 (1976).

  4. 4

    Schwacha, A. & Kleckner, N. Cell 83, 783–791 (1995).

  5. 5

    Duckett, D.R., Giraud-Panis, M.-J.E. & Lilley, D.M.J. J. Mol. Biol. 246, 95–107 (1995).

  6. 6

    Pöhler, J.R.G., Giraud-Panis, M.-J.E. & Lilley, D.M.J. J. Mol. Biol. 260, 678–696 (1996).

  7. 7

    White, M.F. & Lilley, D.M.J. J. Mol. Biol. 257, 330–341 (1996).

  8. 8

    Studier, F.W. Virology 39, 562–574 (1969).

  9. 9

    de Massey, B., Studier, F.W., Dorgai, L., Appelbaum, F. & Weisberg, R.A. Cold Spring Harbor Symp. Quant. Biol. 49, 715–726 (1984).

  10. 10

    de Massey, B., Weisberg, R.A. & Studier, F.W. J. Mol. Biol. 193, 359–376 (1987).

  11. 11

    Dickie, P., McFadden, G. & Morgan, A.R. J. Biol. Chem. 262, 14826–14836 (1987).

  12. 12

    Kerr, C. & Sadowski, P.D. Virology 65, 281–285 (1975).

  13. 13

    Powling, A. & Knippers, R. Mol. Gen. Genet. 149, 63–71 (1976).

  14. 14

    Tsujimoto, Y. & Ogawa, H. J. Mol. Biol. 125, 255–273 (1978).

  15. 15

    Parkinson, M.J. & Lilley, D.M.J. J. Mol. Biol. 270, 169–178 (1997).

  16. 16

    Ariyoshi, M. et al. Cell 78, 1063–1072 (1994).

  17. 17

    Raaijmakers, H. et al. EMBO J. 18, 1447–1458 (1999).

  18. 18

    Terwilliger, T.C. & Berendzen, J. Acta Crystallogr. D 53, 571–579 (1997).

  19. 19

    Collaborative Computational Project, Number 4. Acta Crystallogr. D 50, 760–763 (1994).

  20. 20

    Rafferty, J.B. et al. Science 274, 415–421 (1996).

  21. 21

    Guo, F., Gopaul, D.N. & Van Duyne, G.D. Nature 389, 40–46 (1997).

  22. 22

    Parkinson, M J., Pöhler, J.R.G. & Lilley, D.M.J. Nucleic Acids Res. 27, 682–689 (1999).

  23. 23

    Newman, M. et al. EMBO J. 17, 5466–5476 (1998).

  24. 24

    Winkler, F.K. et al. EMBO J. 12, 1781–1795 (1993).

  25. 25

    Kim, Y., Grable, J.C., Love, R., Greene, P.J. & Rosenberg, J.M. Science 249, 1307–1309 (1990).

  26. 26

    Wah, D.A., Hirsch, J.A., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Nature 388, 97–100 (1997).

  27. 27

    Pingould, A. & Jeltsch, A. Eur. J. Biochem. 246, 1–22 (1997).

  28. 28

    Lee, J., Jayaram, M. & Grainge, I. EMBO J. 18, 784–791 (1999).

  29. 29

    Holm, L. & Sander, C. J. Mol. Biol. 233, 123–138 (1993).

  30. 30

    Deibert, M., Grazulis, S., Janulaitis, A., Siksnys, V. & Huber, R. EMBO J. 18, 5805–5816 (1999).

  31. 31

    Bozic, D., Grazulis, S., Siksnys, V. & Huber, R. J. Mol. Biol. 255, 176–186 (1996).

  32. 32

    Hickman, A.B. et al. Mol. Cell. 5, 1025–1034 (2000).

  33. 33

    Hargreaves, D. et al. Nature Struct. Biol. 5, 441–445 (1998).

  34. 34

    Eichman, B.F., Vargason, J.M., Mooers, B.H.M. & Ho, P.S. Proc. Natl. Acad. Sci. USA 97, 3971–3976 (2000).

  35. 35

    Giraud-Panis, M.-J.E. & Lilley, D.M.J. J. Biol. Chem. 271, 33148–33155 (1996).

  36. 36

    Kvaratskhelia, M., Wardleworth, B.N., Norman, D.G. & White, M.F. J. Biol. Chem. 275, 25540–25546 (2000).

  37. 37

    Ban, C. & Yang, W. EMBO J. 17, 1526–1534 (1998).

  38. 38

    Kovall, R. & Matthews, B.W. Science 277, 1824–1827 (1997).

  39. 39

    Lilley, D.M.J. & White, M.F. Proc. Natl. Acad. Sci. USA 97, 9351–9353 (2000).

  40. 40

    LeMaster, D.M. & Richards, F.M. Biochemistry 24, 7263–7268 (1985).

  41. 41

    Graber, P. et al. Eur. J. Biochem. 212, 751–755 (1993).

  42. 42

    Leslie, A.G.W. MOSFLM. (MRC Laboratory of Molecular Biology, Cambridge, UK; 1996).

  43. 43

    Jones, T.A., Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. Acta Crystallogr. A 47, 110–119 (1991).

  44. 44

    Brünger, A.T. X-PLOR 3.1: a system for crystallography and NMR (Yale University Press, New Haven, Conneticut; 1993).

  45. 45

    Brünger, A.T. et al. Acta Crystallogr. D 54, 905–921 (1998).

  46. 46

    Duckett, D.R. et al. Cell 55, 79–89 (1988).

  47. 47

    Laskwoski, R.A., MacArthur, M.W., Moss, D.W. & Thornton, J.M. J. Appl. Crystallogr. 26, 283–291 (1993).

  48. 48

    Christopher, J.A. The Spock Homepage: http://quorum.tamu.edu/spock/ (1998).

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Acknowledgements

We would like to thank G. Leonard, S. Azrt, R. Ravelli, and S. McSweeney for help with data collection at the European Synchrotron Radiation Facility and the staff of the Daresbury SRS for assistance with preliminary studies. We are grateful to the Wellcome Trust and the Cancer Research Campaign for financial support and for facilities provided by the BBSRC-funded North of England Structural Biology Centre (NESBIC).

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Correspondence to Simon E.V. Phillips.

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Hadden, J., Convery, M., Déclais, A. et al. Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I. Nat Struct Mol Biol 8, 62–67 (2001). https://doi.org/10.1038/83067

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