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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References
Dryden, D.T.F., Murray, N. & Rao, D.N. Nucleoside triphosphate-dependent restriction enzymes. Nucleic Acids Res. 29, 3728–3741 (2001).
Labrie, S.J., Samson, J.E. & Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327 (2010).
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).
Meselson, M. & Yuan, R. DNA restriction enzyme from E. coli. Nature 217, 1110–1114 (1968).
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).
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).
Murray, N.E. type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol. Mol. Biol. Rev. 64, 412–434 (2000).
Rao, D.N., Dryden, D.T. & Bheemanaik, S. Type III restriction-modification enzymes: a historical perspective. Nucleic Acids Res. 42, 45–55 (2014).
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).
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).
Halford, S.E., Welsh, A.J. & Szczelkun, M.D. Enzyme-mediated DNA looping. Annu. Rev. Biophys. Biomol. Struct. 33, 1–24 (2004).
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).
Kennaway, C.K. et al. Structure and operation of the DNA-translocating type I DNA restriction enzymes. Genes Dev. 26, 92–104 (2012).
Thomä, N.H. et al. Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat. Struct. Mol. Biol. 12, 350–356 (2005).
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).
Narlikar, G.J., Sundaramoorthy, R. & Owen-Hughes, T. Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes. Cell 154, 490–503 (2013).
Wollmann, P. et al. Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP. Nature 475, 403–407 (2011).
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).
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).
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).
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).
Shen, B.W. et al. Characterization and crystal structure of the type IIG restriction endonuclease RM.BpuSI. Nucleic Acids Res. 39, 8223–8236 (2011).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Seidel, R. et al. Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. EMBO J. 24, 4188–4197 (2005).
Liu, L.F. & Wang, J.C. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84, 7024–7027 (1987).
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).
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).
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).
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).
Park, J. et al. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps. Cell 142, 544–555 (2010).
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).
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).
Dillingham, M.S. & Kowalczykowski, S.C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008).
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).
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).
Levy, A. et al. CRISPR adaptation biases explain preferences for acquisition of foreign DNA. Nature 520, 505–510 (2015).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
LeMaster, D.M. & Richards, F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry 27, 142–150 (1988).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
French, S. & Wilson, K. On the treatment of negative intensity observations. Acta Crystallogr. A 34, 517–525 (1978).
Read, R.J. & McCoy, A.J. Using SAD data in Phaser. Acta Crystallogr. D Biol. Crystallogr. 67, 338–344 (2011).
Cowtan, K.D. & Zhang, K.Y.J. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).
Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Afonine, P.V. et al. Joint X-ray and neutron refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 66, 1153–1163 (2010).
Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).
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).
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).
Lionnet, T. et al. Magnetic trap construction. Cold Spring Harb. Protoc. 2012, 133–138 (2012).
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).
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).
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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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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DOI: https://doi.org/10.1038/nchembio.1926
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