SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions |
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Authors: | E A Fedotova A S Protsenko M V Zakharova N V Lavrova A V Alekseevsky T S Oretskaya A S Karyagina A S Solonin E A Kubareva |
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Institution: | (1) Intercollegiate Faculty of Biotechnology, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland;(2) Department of Microbiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland;(3) Intercollegiate Faculty of Biotechnology, Department of Molecular and Cellular Biology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland;(4) Present address: Department of Pathology, University of Alabama, Birmingham, AL, USA |
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Abstract: | 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 methyl-transferase 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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Keywords: | regulation in restriction-modification systems (cytosine-5)-DNA methyltransferase DNA-protein interactions |
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