Isolation and Characterization of Biochemical Properties of DNA Methyltransferase FauIA Modifying the Second Cytosine in the Nonpalindromic Sequence 5´-CCCGC-3´

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V. A. Chernukhin^1*, Yu. G. Kashirina¹, K. S. Sukhanova¹, M. A. Abdurashitov², D. A. Gonchar², and S. Kh. Degtyarev²

¹NPO SibEnzyme, ul. Timakova 9, 630117 Novosibirsk, Russia; E-mail: Walera@sibenzyme.ru

²Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Sciences, 630117 Novosibirsk, Russia

^* To whom correspondence should be addressed.

Received May 19, 2004; Revision received September 8, 2004

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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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KEY WORDS: Flavobacterium aquatile, DNA methyltransferases, restriction-modification systems, enzyme kinetics

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