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Crystal structure of T4 endonuclease VII resolving a Holliday junction

Abstract

Holliday proposed a four-way DNA junction as an intermediate in homologous recombination1, and such Holliday junctions have since been identified as a central component in DNA recombination and repair2. Phage T4 endonuclease VII (endo VII) was the first enzyme shown to resolve Holliday junctions into duplex DNAs by introducing symmetrical nicks in equivalent strands3. Several Holliday junction resolvases have since been characterized4, but an atomic structure of a resolvase complex with a Holliday junction remained elusive. Here we report the crystal structure of an inactive T4 endo VII(N62D) complexed with an immobile four-way junction with alternating arm lengths of 10 and 14 base pairs. The junction is a hybrid of the conventional square-planar and stacked-X conformation. Endo VII protrudes into the junction point from the minor groove side, opening it to a 14 Å × 32 Å parallelogram. This interaction interrupts the coaxial stacking, yet every base pair surrounding the junction remains intact. Additional interactions involve the positively charged protein and DNA phosphate backbones. Each scissile phosphate that is two base pairs from the crossover interacts with a Mg2+ ion in the active site. The similar overall shape and surface charge potential of the Holliday junction resolvases endo VII, RuvC, Ydc2, Hjc and RecU, despite having different folds, active site composition and DNA sequence preference, suggest a conserved binding mode for Holliday junctions.

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Figure 1: Structure of the T4 endo VII(N62D)–Holliday junction complex.
Figure 2: Protein–DNA interactions.
Figure 3: The active site of endo VII.
Figure 4: Structural comparisons of Holliday junction and Holliday junction resolvases.

References

  1. Holliday, R. A mechanism for gene conversion in fungi. Genet. Res. 5, 282–304 (1964)

    Article  Google Scholar 

  2. Liu, Y. & West, S. C. Happy Hollidays: 40th anniversary of the Holliday junction. Nature Rev. Mol. Cell Biol. 5, 937–944 (2004)

    Article  CAS  Google Scholar 

  3. Mizuuchi, K., Kemper, B., Hays, J. & Weisberg, R. A. T4 endonuclease VII cleaves Holliday structures. Cell 29, 357–365 (1982)

    Article  CAS  Google Scholar 

  4. Lilley, D. M. & White, M. F. The junction-resolving enzymes. Nature Rev. Mol. Cell Biol. 2, 433–443 (2001)

    Article  CAS  Google Scholar 

  5. Kemper, B. & Janz, E. Function of gene 49 of bacteriophage T4. I. Isolation and biochemical characterization of very fast-sedimenting DNA. J. Virol. 18, 992–999 (1976)

    Article  CAS  Google Scholar 

  6. Kemper, B. & Brown, D. T. Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA. J. Virol. 18, 1000–1015 (1976)

    Article  CAS  Google Scholar 

  7. Solaro, P. C., Birkenkamp, K., Pfeiffer, P. & Kemper, B. Endonuclease VII of phage T4 triggers mismatch correction in vitro . J. Mol. Biol. 230, 868–877 (1993)

    Article  CAS  Google Scholar 

  8. Raaijmakers, H. et al. X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture. EMBO J. 18, 1447–1458 (1999)

    Article  CAS  Google Scholar 

  9. Raaijmakers, H., Tõrö, I., Birkenbihl, R., Kemper, B. & Suck, D. Conformational flexibility in T4 endonuclease VII revealed by crystallography: implications for substrate binding and cleavage. J. Mol. Biol. 308, 311–323 (2001)

    Article  CAS  Google Scholar 

  10. Duckett, D. R. et al. The structure of the Holliday junction, and its resolution. Cell 55, 79–89 (1988)

    Article  CAS  Google Scholar 

  11. Lilley, D. M. Structures of helical junctions in nucleic acids. Q. Rev. Biophys. 33, 109–159 (2000)

    Article  CAS  Google Scholar 

  12. Pöhler, J. R., Giraud-Panis, M. J. & Lilley, D. M. T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme. J. Mol. Biol. 260, 678–696 (1996)

    Article  Google Scholar 

  13. Giraud-Panis, M. J. & Lilley, D. M. Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII. EMBO J. 16, 2528–2534 (1997)

    Article  CAS  Google Scholar 

  14. Giraud-Panis, M. J. & Lilley, D. M. T4 endonuclease VII. Importance of a histidine-aspartate cluster within the zinc-binding domain. J. Biol. Chem. 271, 33148–33155 (1996)

    Article  CAS  Google Scholar 

  15. Golz, S., Christoph, A., Birkenkamp-Demtröder, K. & Kemper, B. Identification of amino acids of endonuclease VII essential for binding and cleavage of cruciform DNA. Eur. J. Biochem. 245, 573–580 (1997)

    Article  CAS  Google Scholar 

  16. Scholz, S. R. et al. Experimental evidence for a ββα-Me-finger nuclease motif to represent the active site of the caspase-activated DNase. Biochemistry 42, 9288–9294 (2003)

    Article  CAS  Google Scholar 

  17. Yang, W., Lee, J. Y. & Nowotny, M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Mol. Cell 22, 5–13 (2006)

    Article  CAS  Google Scholar 

  18. Stoddard, B. L. Homing endonuclease structure and function. Q. Rev. Biophys. 38, 49–95 (2005)

    Article  CAS  Google Scholar 

  19. Khuu, P. A., Voth, A. R., Hays, F. A. & Ho, P. S. The stacked-X DNA Holliday junction and protein recognition. J. Mol. Recognit. 19, 234–242 (2006)

    Article  CAS  Google Scholar 

  20. Hargreaves, D. et al. Crystal structure of E. coli RuvA with bound DNA Holliday junction at 6 Å resolution. Nature Struct. Biol. 5, 441–446 (1998)

    Article  CAS  Google Scholar 

  21. Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H. & Morikawa, K. Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer. Proc. Natl Acad. Sci. USA 97, 8257–8262 (2000)

    Article  ADS  CAS  Google Scholar 

  22. Gopaul, D. N., Guo, F. & Van Duyne, G. D. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175–4187 (1998)

    Article  CAS  Google Scholar 

  23. Conway, A. B., Chen, Y. & Rice, P. A. Structural plasticity of the Flp–Holliday junction complex. J. Mol. Biol. 326, 425–434 (2003)

    Article  CAS  Google Scholar 

  24. Biswas, T. et al. A structural basis for allosteric control of DNA recombination by λ integrase. Nature 435, 1059–1066 (2005)

    Article  ADS  CAS  Google Scholar 

  25. Hadden, J. M., Déclais, A.-C., Carr, S. B., Lilley, D. M. J. & Phillips, E. V. The structural basis of Holliday junction resolution by T7 endonuclease I. Nature advance online publication. doi: 10.1038/nature06158 (2007)

  26. Déclais, A. C., Hadden, J., Phillips, S. E. & Lilley, D. M. The active site of the junction-resolving enzyme T7 endonuclease I. J. Mol. Biol. 307, 1145–1158 (2001)

    Article  Google Scholar 

  27. Ariyoshi, M. et al. Atomic structure of the RuvC resolvase: a Holliday junction-specific endonuclease from E. coli . Cell 78, 1063–1072 (1994)

    Article  CAS  Google Scholar 

  28. Bond, C. S., Kvaratskhelia, M., Richard, D., White, M. F. & Hunter, W. N. Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus . Proc. Natl Acad. Sci. USA 98, 5509–5514 (2001)

    Article  ADS  CAS  Google Scholar 

  29. Ceschini, S. et al. Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2. EMBO J. 20, 6601–6611 (2001)

    Article  CAS  Google Scholar 

  30. McGregor, N. et al. The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage. Structure 13, 1341–1351 (2005)

    Article  CAS  Google Scholar 

  31. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

  32. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  33. The. CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  34. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  37. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004)

    Article  Google Scholar 

  38. Lavery, R. & Sklenar, H. Defining the structure of irregular nucleic acids: conventions and principles. J. Biomol. Struct. Dyn. 6, 655–667 (1989)

    Article  CAS  Google Scholar 

  39. Luscombe, N. M., Laskowski, R. A. & Thornton, J. M. NUCPLOT: a program to generate schematic diagrams of protein–nucleic acid interactions. Nucleic Acids Res. 25, 4940–4945 (1997)

    Article  CAS  Google Scholar 

  40. Kleywegt, G. J. Use of non-crystallographic symmetry in protein structure refinement. Acta Crystallogr. D 52, 842–857 (1996)

    Article  CAS  Google Scholar 

  41. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Leahy and M. Gellert for critical reading of the manuscript, S. Ramon-Maiques for collecting the diffracting data of the endo VII–heteroduplex complex, and D. M. Lilley for background reading materials. C.B. thanks J. Basquin, E. Ennifar and C. Sauter for help with crystallization and data collection, and M. Nowotny and J. Y. Lee for help with manuscript preparation. This research was supported by EMBL, Deutsche Forschungsgemeinschaft and the Intramural Research Program of NIDDK, NIH.

Author Contributions C.B. carried out all experiments. The project was initiated at EMBL and finished at NIH. All authors contributed to experimental design, interpretation and manuscript preparation.

Atomic coordinates and structure factors of the endo VII–DNA complexes have been deposited in the Protein Data Bank. The accession codes are 2QNC and 2QNF for the Holliday junction and the heteroduplex complex, respectively.

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Correspondence to Wei Yang or Dietrich Suck.

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Biertümpfel, C., Yang, W. & Suck, D. Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449, 616–620 (2007). https://doi.org/10.1038/nature06152

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