Mutational Analysis of the Catalytic Domain of the Murine Dnmt3a DNA-(cytosine C5)-methyltransferase Humaira Gowher 1 , Panida Loutchanwoot 1,2 , Olga Vorobjeva 3 Vikas Handa 2 , Renata Z. Jurkowska 1 , Tomasz P. Jurkowski 1 and Albert Jeltsch 1 * 1 International University Bremen, Biochemistry, School of Engineering and Science Campus Ring 1, 28759 Bremen Germany 2 Institut fu ¨r Biochemie, FB 08 Heinrich-Buff-Ring 58 Justus-Liebig-Universita ¨t Giessen, 35392 Giessen Germany 3 A.N. Belozersky Institute of Physico-Chemical Biology Moscow State University Moscow 119899, Russian Federation On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein. q 2006 Elsevier Ltd. All rights reserved. Keywords: DNA methyltransferase; enzyme mechanism; site-directed mutagenesis; epigenetics; methylcytosine *Corresponding author Introduction In mammals, DNA methylation occurs at cyto- sine residues mainly within CG dinucleotides which are modified in a cell type specific pat- tern. 1–4 This methylation is established and main- tained by DNA methyltransferases (Dnmts), which transfer a methyl group to the 5-position of cytosine using S-adenosyl-L-methionine (AdoMet) as the donor. 1,2,5,6 DNA methylation is essential for mammalian development and plays crucial roles in various biological processes. It is involved in differentiation, regulation of gene expression, regu- lation of the chromatin structure, DNA replication, parental imprinting, and X-chromosome inacti- vation. 2–4,7 Erroneous methylation leads to carcino- genesis and other diseases. 8–10 There are four mammalian DNA methyltransferases (MTases) known. 1,11,12 The Dnmt1 enzyme prefers hemi- methylated substrates over unmethylated ones and functions as maintenance methyltransferase. 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Abbreviations used: AdoMet, S-adenosyl-L-methion- ine; Dnmt, DNA methyltransferase; MTase, methyltrans- ferase; TRD, target recognition domain; CD, catalytic domain. E-mail address of the corresponding author: a.jeltsch@iu-bremen.de doi:10.1016/j.jmb.2006.01.035 J. Mol. Biol. (2006) 357, 928–941