A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71？kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. 1. Introduction Restriction enzymes recognizing unique DNA sequences are indispensable tools in molecular biology. Among three different types of restriction enzymes that have been described, type II endonucleases are the most useful enzymes for gene cloning because they recognize 4–8？bp sequences and cleave the DNA within or in close proximity to the recognition site [1]. More than 3500 different type II restriction endonucleases with 240 unique specificities have been currently identified [2]. Restriction endonucleases working with methyltransferases form the basis of RE (restriction modification) systems of prokaryotes to protect them against the invasion of foreign DNA [3]. Type II endonucleases consist of structurally and mechanistically diverse subclasses of enzymes, each with a different mechanism of sequence recognition, DNA cleavage, and organization of quaternary structure [4]. The orthodox type IIP enzymes are the most studied and include the majority of enzymes such as EcoRI, EcoRV, and BamHI commonly used in molecular biology [5]. These enzymes typically exist in solution as a homodimer, bind to a symmetrical palindromic 4–8？bp sequence as a dimer, and cleave phosphodiester bonds on both strands of DNA within the
References
[1]
A. Pingoud, M. Fuxreiter, V. Pingoud, and W. Wende, “Type II restriction endonucleases: structure and mechanism,” Cellular and Molecular Life Sciences, vol. 62, no. 6, pp. 685–707, 2005.
[2]
R. J. Roberts, T. Vincze, J. P. Posfai, and D. Macelis, “REBASE: restriction enzymes and methyltransferases,” Nucleic Acids Research, vol. 31, no. 1, pp. 418–420, 2003.
[3]
T. A. Bickle and D. H. Kruger, “Biology of DNA restriction,” Microbiological Reviews, vol. 57, no. 2, pp. 434–450, 1993.
[4]
M. Mucke, D. H. Kruger, and M. Reuter, “Diversity of Type II restriction endonucleases that require two DNA recognition sites,” Nucleic Acids Research, vol. 31, no. 21, pp. 6079–6084, 2003.
[5]
J. J. Perona, “Type II restriction endonucleases,” Methods, vol. 28, no. 3, pp. 353–364, 2002.
[6]
Y. Kim, J. C. Grable, R. Love, P. J. Greene, and J. M. Rosenberg, “Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing,” Science, vol. 249, no. 4974, pp. 1307–1309, 1990.
[7]
M. Newman, T. Strzelecka, L. F. Dorner, I. Schildkraut, and A. K. Aggarwal, “Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding,” Science, vol. 269, no. 5224, pp. 656–663, 1995.
[8]
A. Pingoud and A. Jeltsch, “Structure and function of type II restriction endonucleases,” Nucleic Acids Research, vol. 29, no. 18, pp. 3705–3727, 2001.
[9]
A. J. Bath, S. E. Milsom, N. A. Gormley, and S. E. Halford, “Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA,” The Journal of Biological Chemistry, vol. 277, no. 6, pp. 4024–4033, 2002.
[10]
D. A. Wah, J. A. Hirsch, L. F. Dorner, I. Schildkraut, and A. K. Aggarwal, “Structure of the multimodular endonuclease FokI bound to DNA,” Nature, vol. 388, no. 6637, pp. 97–100, 1997.
[11]
é. S. Vanamee, S. Santagata, and A. K. Aggarwal, “FokI requires two specific DNA sites for cleavage,” Journal of Molecular Biology, vol. 309, no. 1, pp. 69–78, 2001.
[12]
D. H. Krüger, G. J. Barcak, M. Reuter, and H. O. Smith, “EcoRII can be activated to cleave refractory DNA recognition sites,” Nucleic Acids Research, vol. 16, no. 9, pp. 3997–4008, 1988.
[13]
G. Tamulaitis, G. Sasnauskas, M. Mucke, and V. Siksnys, “Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease,” Journal of Molecular Biology, vol. 358, no. 2, pp. 406–419, 2006.
[14]
M. Conrad and M. D. Topal, “DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, no. 24, pp. 9707–9711, 1989.
[15]
D. Bozic, S. Grazulis, V. Siksnys, and R. Huber, “Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 ？ resolution,” Journal of Molecular Biology, vol. 255, no. 1, pp. 176–186, 1996.
[16]
M. Deibert, S. Grazulis, G. Sasnauskas, V. Siksnys, and R. Huber, “Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA,” Nature Structural Biology, vol. 7, no. 9, pp. 792–799, 2000.
[17]
M. Zaremba, G. Sasnauskas, C. Urbanke, and V. Siksnys, “Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations,” Journal of Molecular Biology, vol. 348, no. 2, pp. 459–478, 2005.
[18]
Y. Xu, K. D. Lunnen, and H. Kong, “Engineering a nicking endonuclease N.Alwl by domain swapping,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 23, pp. 12990–12995, 2001.
[19]
A. Joneja and X. Huang, “Linear nicking endonuclease-mediated strand-displacement DNA amplification,” Analytical Biochemistry, vol. 414, no. 1, pp. 58–69, 2011.
[20]
M. Kataoka, K. Ueda, T. Kudo, T. Seki, and T. Yoshida, “Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces,” FEMS Microbiology Letters, vol. 151, no. 2, pp. 249–255, 1997.
[21]
T. Kieser, M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood, Practical Streptomyces Genetics, John Innes Foundation, Norwich, UK, 2000.
[22]
S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, “Basic local alignment search tool,” Journal of Molecular Biology, vol. 215, no. 3, pp. 403–410, 1990.
[23]
D. J. Wright, W. E. Jack, and P. Modrich, “The kinetic mechanism of EcoRI endonuclease,” The Journal of Biological Chemistry, vol. 274, no. 45, pp. 31896–31902, 1999.
[24]
P. Hensley, G. Nardone, J. G. Chirikjian, and M. E. Wastney, “The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease,” The Journal of Biological Chemistry, vol. 265, no. 25, pp. 15300–15307, 1990.
[25]
L. S. Higgins, C. Besnier, and H. Kong, “The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases Mlyl and Plel,” Nucleic Acids Research, vol. 29, no. 12, pp. 2492–2501, 2001.
[26]
L. Li, L. P. Wu, and S. Chandrasegaran, “Functional domains in Fok I restriction endonuclease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 10, pp. 4275–4279, 1992.
[27]
P. Modrich and D. Zabel, “EcoRI endonuclease. Physical and catalytic properties of the homogeneous enzyme,” The Journal of Biological Chemistry, vol. 251, no. 19, pp. 5866–5874, 1976.
[28]
T. R. Gingeras, L. Greenough, I. Schildkraut, and R. J. Roberts, “Two new restriction endonucleases from Proteus vulgaris,” Nucleic Acids Research, vol. 9, no. 18, pp. 4525–4536, 1981.
[29]
T. F. Meyer and K. Geider, “Bacteriophage fd gene II-protein. II. Specific cleavage and relaxation of supercoiled RF from filamentous phages,” The Journal of Biological Chemistry, vol. 254, no. 24, pp. 12642–12646, 1979.
[30]
T.-L. Chang, M. G. Kramer, R. A. Ansari, and S. A. Khan, “Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication,” The Journal of Biological Chemistry, vol. 275, no. 18, pp. 13529–13534, 2000.
[31]
E. Yokoyama, Y. Matsuzaki, K. Doi, and S. Ogata, “Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921,” FEMS Microbiology Letters, vol. 169, no. 1, pp. 103–109, 1998.
[32]
K. G. Au, K. Welsh, and P. Modrich, “Initiation of methyl-directed mismatch repair,” The Journal of Biological Chemistry, vol. 267, no. 17, pp. 12142–12148, 1992.
[33]
S. Niu, Q. Li, L. Qu, and W. Wang, “Nicking endonuclease and target recycles signal amplification assisted quantum dots for fluorescence detection of DNA,” Analytica Chimica Acta, vol. 680, no. 1-2, pp. 54–58, 2010.
