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J. Biol. Chem., Vol. 278, Issue 43, 41837-41848, October 24, 2003

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Identification and Mutational Analysis of Mg²⁺ Binding Site in EcoP15I DNA Methyltransferase

INVOLVEMENT IN TARGET BASE EVERSION^*

Pradeep Bist and Desirazu N. Rao

From the Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

EcoP15I DNA methyltransferase catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the N⁶ position of the second adenine within the double-stranded DNA sequence 5'-CAGCAG-3'. To achieve catalysis, the enzyme requires a magnesium ion. Binding of magnesium to the enzyme induces significant conformational changes as monitored by circular dichroism spectroscopy. EcoP15I DNA methyltransferase was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 8.0. The inactivated enzyme was cleaved into two fragments with molecular masses of 36 and 35 kDa. Using this affinity cleavage assay, we have located the magnesium binding-like motif to amino acids 355–377 of EcoP15I DNA methyltransferase. Sequence homology comparisons between EcoP15I DNA methyltransferase and other restriction endonucleases allowed us to identify a PD(X)_n(D/E)XK-like sequence as the putative magnesium ion binding site. Point mutations generated in this region were analyzed for their role in methyltransferase activity, metal coordination, and substrate binding. Although the mutant methyltransferases bind DNA and S-adenosyl-L-methionine as well as the wild-type enzyme does, they are inactive primarily because of their inability to flip the target base. Collectively, these data are consistent with the fact that acidic amino acid residues of the region 355–377 in EcoP15I DNA methyltransferase are important for the critical positioning of magnesium ions for catalysis. This is the first example of metal-dependent function of a DNA methyltransferase. These findings provide impetus for exploring the role(s) of metal ions in the structure and function of DNA methyltransferases.

Received for publication, July 2, 2003 , and in revised form, July 30, 2003.

^* This research was supported in part by a grant from Council of Scientific and Industrial Research, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Department of Biotechnology PostDoctoral Fellowship.

To whom correspondence should be addressed. Tel.: 91-80-293-2538; Fax: 91-80-360-0814; E-mail: dnrao{at}biochem.iisc.ernet.in.

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