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Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Abstract

Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme−DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.

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Figure 1: Architecture of the modular Type ISP RM enzymes.
Figure 2: DNA target recognition by LlaBIII.
Figure 3: Architecture and upstream positioning of the ATPase domains.
Figure 4: Loop-independent translocation.
Figure 5: Single-cleavage-event mapping.
Figure 6: Model for loop-independent DNA translocation and extensive nucleolytic DNA processing.

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References

  1. Dryden, D.T.F., Murray, N. & Rao, D.N. Nucleoside triphosphate-dependent restriction enzymes. Nucleic Acids Res. 29, 3728–3741 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Labrie, S.J., Samson, J.E. & Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327 (2010).

    CAS  PubMed  Google Scholar 

  3. Linn, S. & Arber, W. Host specificity of DNA produced by Escherichia coli, X. In vitro restriction of phage fd replicative form. Proc. Natl. Acad. Sci. USA 59, 1300–1306 (1968).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Meselson, M. & Yuan, R. DNA restriction enzyme from E. coli. Nature 217, 1110–1114 (1968).

    CAS  PubMed  Google Scholar 

  5. Loenen, W.A.M., Dryden, D.T.F., Raleigh, E.A. & Wilson, G.G. Type I restriction enzymes and their relatives. Nucleic Acids Res. 42, 20–44 (2014).

    CAS  PubMed  Google Scholar 

  6. Smith, R.M., Diffin, F.M., Savery, N.J., Josephsen, J. & Szczelkun, M.D. DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI. Nucleic Acids Res. 37, 7206–7218 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Murray, N.E. type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol. Mol. Biol. Rev. 64, 412–434 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Rao, D.N., Dryden, D.T. & Bheemanaik, S. Type III restriction-modification enzymes: a historical perspective. Nucleic Acids Res. 42, 45–55 (2014).

    CAS  PubMed  Google Scholar 

  9. Dürr, H., Flaus, A., Owen-Hughes, T. & Hopfner, K.-P. Snf2 family ATPases and DExx box helicases: differences and unifying concepts from high-resolution crystal strucures. Nucleic Acids Res. 34, 4160–4167 (2006).

    PubMed  PubMed Central  Google Scholar 

  10. Stanley, L.K. et al. When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I. EMBO J. 25, 2230–2239 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Halford, S.E., Welsh, A.J. & Szczelkun, M.D. Enzyme-mediated DNA looping. Annu. Rev. Biophys. Biomol. Struct. 33, 1–24 (2004).

    CAS  PubMed  Google Scholar 

  12. Schwarz, F.W. et al. The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA. Science 340, 353–356 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kennaway, C.K. et al. Structure and operation of the DNA-translocating type I DNA restriction enzymes. Genes Dev. 26, 92–104 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Thomä, N.H. et al. Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat. Struct. Mol. Biol. 12, 350–356 (2005).

    PubMed  Google Scholar 

  15. Hopfner, K.-P., Gerhold, C.-B., Lakomek, K. & Wollmann, P. Swi2/Snf2 remodelers: hybrid views on hybrid molecular machines. Curr. Opin. Struct. Biol. 22, 225–233 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Narlikar, G.J., Sundaramoorthy, R. & Owen-Hughes, T. Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes. Cell 154, 490–503 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Wollmann, P. et al. Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP. Nature 475, 403–407 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Smith, R.M., Josephsen, J. & Szczelkun, M.D. The single polypeptide restriction-modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops. Nucleic Acids Res. 37, 7219–7230 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Šišáková, E., van Aelst, K., Diffin, F.M. & Szczelkun, M.D. The type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites. Nucleic Acids Res. 41, 1071–1080 (2013).

    PubMed  Google Scholar 

  20. van Aelst, K., Šišáková, E. & Szczelkun, M.D. DNA cleavage by type ISP Restriction-Modification enzymes is initially targeted to the 3′-5′ strand. Nucleic Acids Res. 41, 1081–1090 (2013).

    CAS  PubMed  Google Scholar 

  21. Park, S.Y. et al. Structural characterization of a modification subunit of a putative type I restriction enzyme from Vibrio vulnificus YJ016. Acta Crystallogr. D Biol. Crystallogr. 68, 1570–1577 (2012).

    CAS  PubMed  Google Scholar 

  22. Shen, B.W. et al. Characterization and crystal structure of the type IIG restriction endonuclease RM.BpuSI. Nucleic Acids Res. 39, 8223–8236 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Goedecke, K., Pignot, M., Goody, R.S., Scheidig, A.J. & Weinhold, E. Structure of the N6-adenine DNA methyltransferase M•TaqI in complex with DNA and a cofactor analog. Nat. Struct. Biol. 8, 121–125 (2001).

    CAS  PubMed  Google Scholar 

  24. Kim, J.S. et al. Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications. Proc. Natl. Acad. Sci. USA 102, 3248–3253 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Calisto, B.M. et al. Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium. J. Mol. Biol. 351, 749–762 (2005).

    CAS  PubMed  Google Scholar 

  26. Velankar, S.S., Soultanas, P., Dillingham, M.S., Subramanya, H.S. & Wigley, D.B. Crystal structures of complexes of PcrA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84 (1999).

    CAS  PubMed  Google Scholar 

  27. Lee, J.Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Büttner, K., Nehring, S. & Hopfner, K.-P. Structural basis for DNA duplex separation by a superfamily-2 helicase. Nat. Struct. Mol. Biol. 14, 647–652 (2007).

    PubMed  Google Scholar 

  29. Saikrishnan, K., Powell, B., Cook, N.J., Webb, M.R. & Wigley, D.B. Mechanistic basis of 5′-3′ translocation in SF1B helicases. Cell 137, 849–859 (2009).

    CAS  PubMed  Google Scholar 

  30. Gu, M. & Rice, C.M. Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc. Natl. Acad. Sci. USA 107, 521–528 (2010).

    CAS  PubMed  Google Scholar 

  31. Dürr, H., Körner, C., Müller, M., Hickmann, V. & Hopfner, K.P. X-ray structures of the Sulfolobus solfataricus SWI2/SNF2 ATPase core and its complex with DNA. Cell 121, 363–373 (2005).

    PubMed  Google Scholar 

  32. Ramanathan, S.P. et al. type III restriction enzymes communicate in 1D without looping between their target sites. Proc. Natl. Acad. Sci. USA 106, 1748–1753 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Seidel, R. et al. Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. EMBO J. 24, 4188–4197 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Liu, L.F. & Wang, J.C. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84, 7024–7027 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Seidel, R. et al. Real-time observation of DNA translocation by the type I RM enzyme EcoR124I. Nat. Struct. Mol. Biol. 11, 838–843 (2004).

    CAS  PubMed  Google Scholar 

  36. Sisáková, E., Weiserova, M., Dekker, C., Seidel, R. & Szczelkun, M.D. The interrelationship of helicase and nuclease somains during DNA translocation by the molecular motor EcoR124I. J. Mol. Biol. 384, 1273–1286 (2008).

    PubMed  PubMed Central  Google Scholar 

  37. Smith, R.M., Josephsen, L. & Szczelkun, M.D. An Mrr-family nuclease motif in the single polypeptide restriction-modification enzyme LlaGI. Nucleic Acids Res. 37, 7231–7238 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Jankowsky, E., Gross, C.H., Shuman, S. & Pyle, A.M. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125 (2001).

    CAS  PubMed  Google Scholar 

  39. Park, J. et al. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps. Cell 142, 544–555 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Fagerburg, M.V. et al. PcrA-mediated disruption of RecA nucleoprotein filaments–essential role of the ATPase activity of RecA. Nucleic Acids Res. 40, 8416–8424 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Taylor, A.F. & Smith, G.R. Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli. J. Mol. Biol. 185, 431–443 (1985).

    CAS  PubMed  Google Scholar 

  42. Dillingham, M.S. & Kowalczykowski, S.C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Yeeles, J.T., Cammack, R. & Dillingham, M.S. An iron-sulfur cluster is essential for the binding of broken DNA by AddAB-type helicase-nucleases. J. Biol. Chem. 284, 7746–7755 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Endlich, B. & Linn, S. The DNA restriction endonuclease of Escherichia coli B. II. Futher studies of the structure of DNA intermediates and products. J. Biol. Chem. 260, 5729–5738 (1985).

    CAS  PubMed  Google Scholar 

  45. Levy, A. et al. CRISPR adaptation biases explain preferences for acquisition of foreign DNA. Nature 520, 505–510 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. LeMaster, D.M. & Richards, F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry 27, 142–150 (1988).

    CAS  PubMed  Google Scholar 

  48. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. French, S. & Wilson, K. On the treatment of negative intensity observations. Acta Crystallogr. A 34, 517–525 (1978).

    Google Scholar 

  50. Read, R.J. & McCoy, A.J. Using SAD data in Phaser. Acta Crystallogr. D Biol. Crystallogr. 67, 338–344 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Cowtan, K.D. & Zhang, K.Y.J. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).

    CAS  PubMed  Google Scholar 

  52. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Afonine, P.V. et al. Joint X-ray and neutron refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 66, 1153–1163 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    CAS  PubMed  Google Scholar 

  55. Butterer, A. et al. type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA. Nucleic Acids Res. 42, 5139–5150 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Szczelkun, M.D. et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl. Acad. Sci. USA 111, 9798–9803 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Lionnet, T. et al. Magnetic trap construction. Cold Spring Harb. Protoc. 2012, 133–138 (2012).

    PubMed  Google Scholar 

  58. Jindrova, E., Schmid-Nuoffer, S., Hamburger, F., Janscak, P. & Bickle, T.A. On the DNA cleavage mechanism of type I restriction enzymes. Nucleic Acids Res. 33, 1760–1766 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Chang, A.C. & Cohen, S.N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141–1156 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was funded by the Wellcome Trust-DBT India Alliance (500048-Z-09-Z to K.S.), Wellcome Trust (084086 to M.D.S., K.v.A., F.M.D. and C.P.), DBT India (to M.K.C.), and CSIR India (to N.N.). K.S. acknowledges K. Nagai and his group members at MRC Laboratory of Molecular Biology, Cambridge, UK, for hosting him as an India Alliance visiting scientist during the initial stage of this work. We thank K. Nagai, D. Wigley, L. Passmore and R. Chauhan for their comments on the manuscript. We acknowledge Diamond Light Source (DLS), Oxfordshire, UK, European Synchrotron Radiation Facility (ESRF), Grenoble, France, for access to beamlines, and DBT India for funding the use of BM14 beamline at ESRF.

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M.K.C. and K.S. purified and crystallized protein, collected and processed the diffraction data, and determined the structure; N.N. contributed to purification and crystallization; M.K. contributed to structure determination; K.v.A. performed the triplex-displacement and nick-mapping gel assays; M.D.S. performed the MTM assays; F.M.D. performed the single-cleavage-event mapping assay; C.P. performed SEC-MALS measurements and analysis. M.D.S. and K.S. designed the study, analyzed data and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Mark D Szczelkun or Kayarat Saikrishnan.

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Chand, M., Nirwan, N., Diffin, F. et al. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes. Nat Chem Biol 11, 870–877 (2015). https://doi.org/10.1038/nchembio.1926

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