Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis of sequence-specific Holliday junction cleavage by MOC1

Nature Chemical Biology volume 15, pages 1241–1248 (2019)Cite this article

Subjects

Abstract

The Holliday junction (HJ) is a key intermediate during homologous recombination and DNA double-strand break repair. Timely HJ resolution by resolvases is critical for maintaining genome stability. The mechanisms underlying sequence-specific substrate recognition and cleavage by resolvases remain elusive. The monokaryotic chloroplast 1 protein (MOC1) specifically cleaves four-way DNA junctions in a sequence-specific manner. Here, we report the crystal structures of MOC1 from Zea mays, alone or bound to HJ DNA. MOC1 uses a unique β-hairpin to embrace the DNA junction. A base-recognition motif specifically interacts with the junction center, inducing base flipping and pseudobase-pair formation at the strand-exchanging points. Structures of MOC1 bound to HJ and different metal ions support a two-metal ion catalysis mechanism. Further molecular dynamics simulations and biochemical analyses reveal a communication between specific substrate recognition and metal ion-dependent catalysis. Our study thus provides a mechanism for how a resolvase turns substrate specificity into catalytic efficiency.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: MOC1 cleaves HJ DNA in a sequence-specific manner.
Fig. 2: Structures of ZmMOC1 and its complex with a HJ.
Fig. 3: Interactions between MOC1 and HJ DNA backbones.
Fig. 4: Base-specific recognition.
Fig. 5: The active sites of MOC1 and metal ion coordination.
Fig. 6: The communication between substrate binding and metal ion coordination, as revealed by molecular dynamics simulations.

Similar content being viewed by others

Data availability

Atomic coordinates and structure factors of apo-MOC1, MOC1–HJ–Ca2+, MOC1D115N–HJ–Mg2+ and MOC1H253A–HJ–Mg2+ have been deposited in the Protein Data Bank (PDB) with accession codes 6IS9, 6JRF, 6IS8 and 6JRG, respectively. All data generated and analyzed during this study are included in this published article. Constructs encoding full-length and truncated ZmMOC1 variants are available from the corresponding author upon reasonable request.

References

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

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  4. Connolly, B. et al. Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc. Natl Acad. Sci. USA 88, 6063–6067 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A. & Shinagawa, H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 10, 4381–4389 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Dunderdale, H. J. et al. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature 354, 506–510 (1991).

    CAS  PubMed  Google Scholar 

  7. Komori, K., Sakae, S., Shinagawa, H., Morikawa, K. & Ishino, Y. A holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea. Proc. Natl Acad. Sci. USA 96, 8873–8878 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kleff, S., Kemper, B. & Sternglanz, R. Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease. EMBO J. 11, 699–704 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Symington, L. S. & Kolodner, R. Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions. Proc. Natl Acad. Sci. USA 82, 7247–7251 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. West, S. C., Parsons, C. A. & Picksley, S. M. Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions. J. Biol. Chem. 262, 12752–12758 (1987).

    CAS  PubMed  Google Scholar 

  11. White, M. F. & Lilley, D. M. Characterization of a holliday junction-resolving enzyme from Schizosaccharomyces pombe. Mol. Cell Biol. 17, 6465–6471 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Constantinou, A., Davies, A. A. & West, S. C. Branch migration and holliday junction resolution catalyzed by activities from mammalian cells. Cell 104, 259–268 (2001).

    CAS  PubMed  Google Scholar 

  13. Boddy, M. N. et al. Mus81-Eme1 are essential components of a holliday junction resolvase. Cell 107, 537–548 (2001).

    CAS  PubMed  Google Scholar 

  14. Ip, S. C. et al. Identification of holliday junction resolvases from humans and yeast. Nature 456, 357–361 (2008).

    CAS  PubMed  Google Scholar 

  15. Wyatt, H. D. & West, S. C. Holliday junction resolvases. Cold Spring Harb. Perspect. Biol. 6, a023192 (2014).

    PubMed  PubMed Central  Google Scholar 

  16. Shah, R., Bennett, R. J. & West, S. C. Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro. Cell 79, 853–864 (1994).

    CAS  PubMed  Google Scholar 

  17. White, M. F. & Lilley, D. M. The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae. J. Mol. Biol. 257, 330–341 (1996).

    CAS  PubMed  Google Scholar 

  18. Kobayashi, Y. et al. Holliday junction resolvases mediate chloroplast nucleoid segregation. Science 356, 631–634 (2017).

    CAS  PubMed  Google Scholar 

  19. Chen, L., Shi, K., Yin, Z. & Aihara, H. Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during holliday junction resolution. Nucleic Acids Res. 41, 648–656 (2013).

    CAS  PubMed  Google Scholar 

  20. Gorecka, K. M., Komorowska, W. & Nowotny, M. Crystal structure of RuvC resolvase in complex with holliday junction substrate. Nucleic Acids Res. 41, 9945–9955 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Biertumpfel, C., Yang, W. & Suck, D. Crystal structure of T4 endonuclease VII resolving a holliday junction. Nature 449, 616–620 (2007).

    PubMed  Google Scholar 

  22. Hadden, J. M., Declais, A. C., Carr, S. B., Lilley, D. M. & Phillips, S. E. The structural basis of holliday junction resolution by T7 endonuclease I. Nature 449, 621–624 (2007).

    CAS  PubMed  Google Scholar 

  23. Nowotny, M., Gaidamakov, S. A., Crouch, R. J. & Yang, W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121, 1005–1016 (2005).

    CAS  PubMed  Google Scholar 

  24. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Nowotny, M. & Yang, W. Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release. EMBO J. 25, 1924–1933 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Majorek, K. A. et al. The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification. Nucleic Acids Res. 42, 4160–4179 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    CAS  PubMed  Google Scholar 

  28. 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).

    PubMed  Google Scholar 

  29. Wang, Q. -S et al. Upgrade of macromolecular crystallography beamline BL17U1 at SSRF. Nucl. Sci. Tech. 29, 68 (2018).

    Google Scholar 

  30. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    PubMed  Google Scholar 

  31. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

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

    Article  PubMed  Google Scholar 

  34. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  35. Case, D. A. et al. AMBER 2016 (University of California: San Francisco, CA, USA, 2016).

  36. Gordon, J. C. et al. H++: a server for estimating pK(a)s and adding missing hydrogens to macromolecules. Nucleic Acids Res. 33, W368–W371 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Ivani, I. et al. Parmbsc1: a refined force field for DNA simulations. Nat. Methods 13, 55–58 (2016).

    CAS  PubMed  Google Scholar 

  39. Aqvist, J. Ion water interaction potentials derived from free-energy perturbation simulations. J. Phys. Chem. 94, 8021–8024 (1990).

    Google Scholar 

  40. Li, P. F. & Merz, K. M. Taking into account the ion-induced dipole interaction in the nonbonded model of ions. J. Chem. Theory Comput. 10, 289–297 (2014).

    CAS  PubMed  Google Scholar 

  41. Panteva, M. T., Giambasu, G. M. & York, D. M. Comparison of structural, thermodynamic, kinetic and mass transport properties of Mg2+ ion models commonly used in biomolecular simulations. J. Comput. Chem. 36, 970–982 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Panteva, M. T., Giambasu, G. M. & York, D. M. Force field for Mg2+, Mn2+, Zn2+, and Cd2+ ions that have balanced interactions with nucleic acids. J. Phys. Chem. B 119, 15460–15470 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Sponer, J. et al. RNA structural dynamics as captured by molecular simulations: a comprehensive overview. Chem. Rev. 118, 4177–4338 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Li, P. F. & Merz, K. M. Metal ion modeling using classical mechanics. Chem. Rev. 117, 1564–1686 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    CAS  Google Scholar 

  46. Toukmaji, A., Sagui, C., Board, J. & Darden, T. Efficient particle-mesh ewald based approach to fixed and induced dipolar interactions. J. Chem. Phys. 113, 10913–10927 (2000).

    CAS  Google Scholar 

  47. Hoover, W. G. Canonical dynamics - equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985).

    CAS  Google Scholar 

  48. Zhang, Y. H., Feller, S. E., Brooks, B. R. & Pastor, R. W. Computer-simulation of liquid/liquid interfaces. 1. Theory and application to octane/water. J. Chem. Phys. 103, 10252–10266 (1995).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff of BL17B, BL18U1 and BL19U1 beamlines at the National Facility for Protein Science Shanghai (NFPS) and Shanghai Synchrotron Radiation Facility, Shanghai, People’s Republic of China, for assistance with X-ray data collection. We thank H. Yu (Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX) for critical comments on this manuscript and Y. Huang (Fuzhou University, Fujian, China) for assistance with the molecular dynamics simulations. This work is supported by the National Natural Science Foundation of China 31971222 (Z.L.), 21603033 (J.L.), 31370737 (M.H.) and 31670739 (M.H.), and the National Key R&D Program of China 2017YFE0103200 (M.H.).

Author information

Author notes

  1. These authors contributed equally: Huajian Lin, Danping Zhang, Ke Zuo.

Authors and Affiliations

  1. College of Chemistry, Fuzhou University, Fuzhou, China

    Huajian Lin, Danping Zhang, Ke Zuo, Jinyu Li, Mingdong Huang & Zhonghui Lin

  2. College of Biological Science and Engineering, Fuzhou University, Fuzhou, China

    Cai Yuan

Authors
  1. Huajian Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Danping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ke Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cai Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jinyu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mingdong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhonghui Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.L. carried out all the cloning, DNA binding and cleavage experiments, and the protein purification and crystallization of both native and SeMet MOC1. D.Z. carried out the purification and crystallization of MOC1–HJ complexes. H.L. and D.Z. performed the X-ray data collection. C.Y. performed structure refinements. K.Z. carried out the molecular dynamics simulations under the supervision of J.L. M.H. supervised the project and analyzed the data. Z.L. conceived and designed the project, determined the crystal structures and wrote the manuscript with the input of all authors. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jinyu Li, Mingdong Huang or Zhonghui Lin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figs. 1–17

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lin, H., Zhang, D., Zuo, K. et al. Structural basis of sequence-specific Holliday junction cleavage by MOC1. Nat Chem Biol 15, 1241–1248 (2019). https://doi.org/10.1038/s41589-019-0377-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0377-4

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing