Abstract
A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5′-CCCGC-3′) was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33°C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5′-CCCGC-3′. Kinetic analysis revealed K m and catalytic constant values of 0.16 μM and 0.05 min−1, respectively.
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REFERENCES
Dryden, D. T. F. (1999) in S-Adenosyl-methionine-Dependent Methyltransferases: Structures and Functions (Cheng, X., and Blumenthal, R. M., eds.) World Scientific, Singapore, pp. 283–340.
Degtyarev, S. Kh., Kolykhanov, A. A., Rechkunova, N. I., and Dedkov, V. S. (1989) Bioorg. Khim., 15, 130–132.
Degtyarev, S. Kh., Netesova, N. A., Chizhikov, V. E., and Abdurashitov, M. A. (1998) Biol. Chem., 379, 567–568.
Abdurashitov, M. A., Okhapkina, S. S., Netesova, N. A., Golikova, L. N., Gonchar, D. A., and Degtyarev, S. Kh. (2003) Mol. Biol. (Moscow), 37, 619–624.
Sugisaki, H., Yamamoto, K., and Takanami, M. (1991) J. Biol. Chem., 266, 13952–13957.
Bitinaite, J., Maneliene, Z., Menkevicius, S., Klimasauskas, S., Butkus, V., and Janulaitis, A. A. (1992) Nucleic Acids Res., 20, 4981–4985.
Friedrich, T., Fatemi, M., Gowhar, H., Leismann, O., and Jeltch, A. (2000) Biochim. Biophys. Acta, 1480, 145–159.
Kossykh, V. G., Schlagman, S. L., and Hattman, S. M. (1995) J. Biol. Chem., 270, 14389–14393.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press.
Bhattacharya, S. K., and Dubey, A. K. (1999) J. Biol. Chem., 274, 14743–14749.
Wu, J. C., and Santi, D. V. (1987) J. Biol. Chem., 262, 4778–4786.
Gabbara, S., Sheluho, D., and Bhagwat, A. S. (1995) Biochemistry, 34, 8914–8923.
Lee, K. F., Liaw, Y., and Shaw, P. C. (1996) Biochem. J., 314, 321–326.
Leismann, O., Roth, M., Friedrich, T., Wende, W., and Jeltsch, A. (1998) Eur. J. Biochem., 251, 899–906.
Chernukhin, V. A., Golikova, L. N., Gonchar, D. A., Abdurashitov, M. A., Kashirina, Yu. G., Netesova, N. A., and Degtyarev, S. Kh. (2003) Biochemistry (Moscow), 68, 967–975.
Malygin, E. G., Ovechkina, L. G., Zinoviev, V. V., Lindstrom, W. M., and Reich, N. O. (2001) Mol. Biol. (Moscow), 35, 35–44.
Thielking, V., Dubois, S., Eritja, R., and Guschlbauer, W. (1997) Biol. Chem., 378, 407–415.
Reich, N. O., and Mashhoon, N. (1991) Biochemistry, 30, 2933–2939.
Buryanov, Y. I., Zinoviev, V. V., Gorbunov, T. A., Tuzikov, F. V., Rechkunova, N. I., Malygin, E. G., and Batev, A. A. (1988) Gene, 74, 67–69.
Malygin, E. G., Petrov, N. A., Gorbunov, T. A., Kossykh, V. G., and Hattman, S. M. (1997) Nucleic Acids Res., 30, 3880–3885.
Cohen, H. M., Tawfik, D. S., and Griffiths, A. D. (2002) Nucleic Acids Res., 30, 3880–3885.
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Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 829–837.
Original Russian Text Copyright © 2005 by Chernukhin, Kashirina, Sukhanova, Abdurashitov, Gonchar, Degtyarev.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-125, December 19, 2004.
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Chernukhin, V.A., Kashirina, Y.G., Sukhanova, K.S. et al. Isolation and Characterization of Biochemical Properties of DNA Methyltransferase FauIA Modifying the Second Cytosine in the Nonpalindromic Sequence 5′-CCCGC-3′. Biochemistry (Moscow) 70, 685–691 (2005). https://doi.org/10.1007/s10541-005-0169-1
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DOI: https://doi.org/10.1007/s10541-005-0169-1