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.