Abstract
The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector. The transcriptional start sites were mapped as well as putative −10 and −35 regions of the fokIM promoter. Enzyme overproduction was ensured by cloning the fokIM gene under the φ 10 promoter of phage T7. M·FokI was purified using a two-step chromatography procedure. M·FokI is a monomeric protein with a M r=76,000±1,500 under denaturing conditions. It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione. Deletion mutants in the N- and C-terminus of M·FokI were isolated and characterized. The N-terminal derivative (M·FokIN) methylates the adenine residue within the sequence 5′-GGATG-3′, whereas the C-terminal derivative (M·FokIC) modifies the adenine residue within the sequence 5′-CATCC-3′. Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule. M·FokI and its truncated derivatives require S-adenosyl-l-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-l-homocysteine. The K m values for the methyl donor, S-adenosyl-{spl}-methionine are 0.6 µM (M·FokI), 0.4 µM (M·FokIN), and 0.9 µM (M·FokIC) while the Km values for substrate λ DNA are 1.2 nM (M·FokI), 1.4 nM (M·FokIN), and 1.3 nM (M·FokIC).
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References
Sugisaki, H. and Kanazawa, S. (1981) New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI). Gene 16, 73–78.
Landry, D., Looney, M. C., Feehery, G. R., Slatko, B. E., Jack, W. E., Schildkraut, I., and Wilson, G. G. (1989) M·FokI methylates adenine in both strands of its asymmetric recognition sequence. Gene 77, 1–10.
Kita, K., Kotani, H., Sugisaki, H. and Takanami, M. (1989) The FokI restriction-modification system I. Organization and nucleotide sequences of the restriction and modification genes. J. Biol. Chem. 264, 5751–5756.
Looney, M. C., Moran, L. S., Jack, W. E., Feehery, G. R., Benner, J. S., Slatko, B. E., and Wilson, G. G. (1989) Nucleotide sequence of the FokI restriction-modification system: separate strand specificity domains in the methyltransferase. Gene 80, 193–208.
Li, L., Wu, L. P., and Chandrasegaran, S. (1992) Functional domains in FokI restriction endonuclease. Proc. Natl. Acad. Sci. USA 89, 4275–4279.
Posfai, G. and Szybalski, W. (1988) A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M·FokI. Gene 69, 147–151.
Sugisaki, H., Kita, K., and Takanami, M. (1989) The FokI restriction-modification system II. Presence of two domains in FokI methylase responsible for modification of different DNA strands. J. Biol. Chem. 264, 5757–5761.
Labbé, D., Höltke, H. J., and Lau, P. C. K. (1990) Cloning and characterization of two tandemly arranged DNA methyltransferase genes of Neisseria lactamica: an adenine-specific M·NlaIII and a cytosine-type methylase. Mol. Gen. Genet. 224, 101–110.
Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.
Maurizi, M. R., Trisler, P., and Gottesman, S. (1985) Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable. J. Bacteriol. 164, 1124–1135.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring harbor Laboratory, Cold Spring Harbor N. Y.
Tabor, S. and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074–1078.
Caserta, M., Zacharias, W., Nwankwo, D., Wilson, G. G., and Wells, R. D. (1987) Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase. J. Biol. Chem. 262, 4770–4777.
Szomolanyi, E., Kiss, A., and Venetianer, P. (1980) Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli. Gene 10, 219–225.
Belfort, M., Pedersen-Lane, J., West, J., Ehrenman, D., Maley, K, Chu, G. and Maley, F. (1985) Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level. Cell 41, 375–382.
Hinton, D. (1989) Transcript analyses of the uvsX-40–41 region of bacteriophage T4. Changes in the RNA as infection proceeds. J. Biol. Chem. 264, 14432–14439.
Kaczorowski, T. and Sektas, M. (1996) Rapid removal of unincorporated label and proteins from DNA sequencing reactions. Mol. Biotechnol. 5, 177–181.
Schlossman, D. M., Schmid, S. L., Brael, W. A. and Rothman, J. E. (1984) An enzyme that removes clathrin coats: purification of an uncoating ATPase. J. Cell. Biol. 99, 723–733.
Weber, K. and Osborn, M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412.
Lisser, S. and Margalit, H. (1993) Compilation of E. coli mRNA promoter sequences. Nucl. Acids Res. 21, 1507–1516.
Baneyx, F. and Georgiou, G. (1990) In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT. J. Bacteriol. 172, 491–494.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680–685.
Liberek, K., Osipiuk, J., Zylicz, M., Ang, D., Skorko, J., and Georgopoulos, G. (1990) Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication. J. Biol. Chem., 265, 3022–3029.
Wolfes, H., Fliess, A., Winkler, F. and Pingoud, A. (1986) Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases. Eur. J. Biochem. 159, 267–273.
Seeman, N. C., Rosenberg, J. M. and Rich, A. (1976) Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73, 804–808.
Riordan, J. F. (1973) Functional arginyl residues in carboxypeptidase A. Modification with butanedione. Biochemistry-USA 12, 3915–3923.
Wilkinson, G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. 80, 324–332.
Chung, C. H., Waxman, L. and Goldberg, A. L. (1983) Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli. A labile form of the ATP-dependent protease La that inhibits the wild type protease. J. Biol. Chem. 258, 215–221.
Kaczorowski, T. (1991) Characterization of the cloned FokI methyltransferase from Flavobacterium okeanokoites. Ph.D. Thesis, University of Gdansk.
Smith, H. O. and Kelly, S. V. (1984) in Razin, A., Cedar, H., and Riggs, A.D. (eds), DNA Methylation: Biochemistry and Biological Significance, Springer-Verlag, New York, pp. 39–71.
Wilson, G. G. (1991) Organization of restriction-modification systems. Nucl. Acids Res. 19, 2539–2566.
Kaczorowski, T., Skowron, P., and Podhajska, A. J. (1989) Purification and characterization of the FokI restriction endonuclease. Gene 80, 209–216.
Skowron, P., Kaczorowski, T., Tucholski, J., and Podhajska, A.J. (1993) Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by FokI monomer. Gene 125, 1–10.
Sektas, M., Kaczorowski, T., and Podhajska, A. J. (1992) Purification and properties of the MboII, a class-IIS restriction endonuclease. Nucl. Acids Res. 20, 433–438.
Sektas, M., Kaczorowski, T., and Podhajska, A. J. (1995) Interaction of the MboII restriction endonuclease with DNA. Gene 157, 181–185.
Tucholski, J., Skowron, P. M., and Podhajska, A. J. (1995) MmeI, a class-IIS restriction endonuclease: purification and characterization. Gene 157, 87–92.
Hanish, J. and McClelland, M. (1988) Activity of DNA modification and restriction enzymes in KGB, a potassium glutamate buffer. Gene Anal. Techn. 5, 105–107.
Lusk, J. E., Williams, J. P., and Kennedy, E. P. (1968) Magnesium and the growth of Escherichia coli. J. Biol. Chem. 243, 2618–2624.
McClelland, M., Nelson, M., and Cantor, C. R. (1985) Purification of MboII methylase (GAAGmA) from Moraxella bovis: site specific cleavage of DNA at nine and ten base pair sequences. Nucl. Acids Res. 13, 7171–7182.
Sugisaki, H., Yamamoto, K., and Takanami, M. (1991) The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands. J. Biol. Chem. 266, 13952–13957.
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Kaczorowski, T., Sektas, M., Skowron, P. et al. The FokI methyltransferase from Flavobacterium okeanokoites . Mol Biotechnol 13, 1–15 (1999). https://doi.org/10.1385/MB:13:1:1
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DOI: https://doi.org/10.1385/MB:13:1:1