Co-transfections of reporter plasmids and plasmids encoding the catalytic domain 
of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene 
expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved 
PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, 
we checked for their catalytic activity in vitro. Surprisingly, the activity of 
the cysteine variant and of the corresponding full-length Dnmt3a variant is only 
two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme 
variants carrying E-->A, E-->D or E-->Q exchanges of the ENV glutamate are 
catalytically almost inactive, demonstrating that this residue has a central 
function in catalysis. Since the glutamic acid residue contacts the flipped 
base, its main function could be to hold the target base at a position that 
supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants 
form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does 
not. Therefore, covalent complex formation is not essential in the reaction 
mechanism of Dnmt3a. We propose that correct positioning of the flipped base and 
the cofactor and binding to the transition state of methyl group transfer are 
the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl 
group transfer.