Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

Abstract

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5′ half (CAT) and a canonical 3′ half (GTG). DraIII nicks the 3′ canonical half site at a faster rate than the 5′ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through sitedirected mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

Keywords

DraIII restriction endonuclease / fidelity / substrate specificity / star activity / inter-domain interaction / site-directed mutagenesis

References

[1] Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr58: 1948-1954

[2] Chan SH, Opitz L, Higgins L, O’Loane D, Xu SY (2010) Cofactor requirement of HpyAV restriction endonuclease. PLoS ONE5: e9071

[3] Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr50: 760-763

[4] Cowtan KD (1994) DM: an automated procedure for phase improvement by density modification. CCP4 Newslett31: 34-38

[5] Cowtan K (2006) The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr62: 1002-1011

[6] Dikic J, Menges C, Clarke S, Kokkinidis M, Pingoud A, Wende W, Desbiolles P (2012) The rotation-coupled sliding of EcoRV. Nucleic Acids Res40: 4064-4070

[7] Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr60: 2126-2132

[8] Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA109: E2579-E2586

[9] George J, Chirikjian JG (1982) Sequence-specific endonuclease BamHI: relaxation of sequence recognition. Proc Natl Acad Sci USA79: 2432-2436

[10] Giraud-Panis MJ, Duckett DR, Lilley DM (1995) The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII. J Mol Biol252: 596-610

[11] Grosskopf R, Wolf W, Kessler C (1985) Two new restriction endonucleases Drall and DrallI from Deinococcus radiophilus. Nucleic Acids Res13: 1517-1528

[12] Halford SE, Lovelady BM, McCallum SA (1986) Relaxed specificity of the EcoRV estriction endonuclease. Gene41: 173-181

[13] Horton JR, Blumenthal RM, Cheng X (2004) Restriction endonucleases: structure of the conserved catalytic core and the role of metal ions in DNA cleavage. In: Pingoud AM(ed) Restriction endonucleases. Springer, Berlin, pp 361-392

[14] Ibryashkina EM, Zakharova MV, Baskunov VB, Bogdanova ES, Nagornykh MO, Den’mukhamedov MM, Melnik BS, Kolinski A, Gront D, Feder M (2007) Type II restriction endonuclease R. Eco29kI is a member of the GIY-YIG nuclease superfamily. BMC Struct Biol7: 48

[15] Jack WE, Terry BJ, Modrich P (1982) Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence. Proc Natl Acad Sci USA79: 4010-4014

[16] Jakubauskas A, Giedriene J, Bujnicki JM, Janulaitis A (2007) Identification of a single HNH active site in type IIS restriction endonuclease Eco31I. J Mol Biol370: 157-169

[17] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337: 816-821

[18] Kaminska KH, Kawai M, Boniecki M, Kobayashi I, Bujnicki JM (2008) Type II restriction endonuclease R. Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site. BMC Struct Biol8: 48

[19] Ko TP, Liao CC, Ku WY, Chak KF, Yuan HS (1999) The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein. Structure7: 91-102

[20] Kriukiene E (2006) Domain organization and metal ion requirement of the Type IIS restriction endonuclease MnlI. FEBS Lett580: 6115-6122

[21] Lesser DR, Kurpiewski MR, Jen-Jacobson L (1990) The energetic basis of specificity in the EcoRI endonuclease-DNA interaction. Science250: 776-786

[22] Malyguine E, Vannier P, Yot P (1980) Alteration of the specificity of restriction endonucleases in the presence of organic solvents. Gene8: 163-177

[23] Mehta P, Katta K, Krishnaswamy S (2004) HNH family subclassification leads to identification of commonality in the His-Me endonuclease superfamily. Protein Sci13: 295-300

[24] Miyazono K, Watanabe M, Kosinski J, Ishikawa K, Kamo M, Sawasaki T, Nagata K, Bujnicki JM, Endo Y, Tanokura M (2007) Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res35: 1908-1918

[25] Nasri M, Thomas D (1987) Alteration of the specificity of PvuII restriction endonuclease. Nucleic Acids Res15: 7677-7687

[26] Orlowski J, Bujnicki JM (2008) Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses. Nucleic Acids Res36: 3552-3569

[27] Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol276: 307-326

[28] Pommer AJ, Cal S, Keeble AH, Walker D, Evans SJ, Kuhlmann UC, Cooper A, Connolly BA, Hemmings AM, Moore GR (2001) Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9. J Mol Biol314: 735-749

[29] Rau DC, Sidorova NY (2010) Diffusion of the restriction nuclease EcoRI along DNA. J Mol Biol395: 408-416

[30] Roberts RJ (2005) How restriction enzymes became the workhorses of molecular biology. Proc Natl Acad Sci USA102: 5905-5908

[31] Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev S, Dryden DT, Dybvig K (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res31: 1805-1812

[32] Robinson CR, Sligar SG (1995) Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, PvuII, and EcoRV specificity. Proc Natl Acad Sci USA92: 3444-3448

[33] Sapranauskas R, Sasnauskas G, Lagunavicius A, Vilkaitis G, Lubys A, Siksnys V (2000) Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTAresistant nuclease Nuc of Salmonella typhimurium. J Biol Chem275: 30878-30885

[34] Saravanan M, Bujnicki JM, Cymerman IA, Rao DN, Nagaraja V (2004) Type II restriction endonuclease R. KpnI is a member of the HNH nuclease superfamily. Nucleic Acids Res32: 6129-6135

[35] Saravanan M, Vasu K, Ghosh S, Nagaraja V (2007a) Dual role for Zn²⁺ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease. J Biol Chem282: 32320-32326

[36] Saravanan M, Vasu K, Kanakaraj R, Rao DN, Nagaraja V (2007b) R. KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca²⁺ and Mg²⁺. Nucleic Acids Res35: 2777-2786

[37] Shen BW, Heiter DF, Chan SH, Wang H, Xu SY, Morgan RD, Wilson GG, Stoddard BL (2010) Unusual target site disruption by the rarecutting HNH restriction endonuclease PacI. Structure18: 734-743

[38] Sokolowska M, Czapinska H, Bochtler M (2009) Crystal structure of the beta beta alpha-Me type II restriction endonuclease Hpy99I with target DNA. Nucleic Acids Res37: 3799-3810

[39] Viadiu H, Aggarwal AK (2000) Structure of BamHI bound to nonspecific DNA: a model for DNA sliding. Mol Cell5: 889-895

[40] Vincze T, Posfai J, Roberts RJ (2003) NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res31: 3688-3691

[41] Wei H, Therrien C, Blanchard A, Guan S, Zhu Z (2008) The fidelity index provides a systematic quantitation of star activity of DNA restriction endonucleases. Nucleic Acids Res36: e50

[42] Wright DJ, Jack WE, Modrich P (1999) The kinetic mechanism of EcoRI endonuclease. J Biol Chem274: 31896-31902

RIGHTS & PERMISSIONS

2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.