SsoII-Like DNA-Methyltransferase Ecl18kI: Interaction between Regulatory and Methylating Functions

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E. A. Fedotova¹, A. S. Protsenko^2,3, M. V. Zakharova³, N. V. Lavrova⁴, A. V. Alekseevsky⁵, T. S. Oretskaya¹, A. S. Karyagina⁴, A. S. Solonin³, and E. A. Kubareva^5*

¹Chemical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia

²Pushchino State University, pr. Nauki 3, 142290 Pushchino, Moscow Region, Russia

³Skryabin Institute of Biochemistry and Physiology of Microorganisms, pr. Nauki 5, 142290 Pushchino, Moscow Region, Russia

⁴Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, ul. Gamalei 18, 123098 Moscow, Russia

⁵Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3181; E-mail: kubareva@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received May 29, 2008; Revision received June 6, 2008

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The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction–modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Δ(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction–modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions—regulatory and methylating.

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KEY WORDS: regulation in restriction–modification systems, (cytosine-5)-DNA methyltransferase, DNA–protein interactions

DOI: 10.1134/S0006297909010131

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