TY - JOUR
T1 - Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited
T2 - Covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme
AU - Reither, Sabine
AU - Li, Fuyang
AU - Gowher, Humaira
AU - Jeltsch, Albert
N1 - Funding Information:
The gift of a luciferase reporter plasmid by Dr Nicolleta Landsberger (Università degli Studi dell'Insubria, Busto Arsizio, Italy) is gratefully acknowledged. Thanks are due to A. Hermann, M. Roth, V. Pingoud and A. Pingoud for discussions. This work was supported by the DFG (JE 252/1-2, 1-3) and the BMBF BioFuture program.
PY - 2003/6/13
Y1 - 2003/6/13
N2 - 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.
AB - 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.
KW - 5-methylcytosine
KW - Covalent complex
KW - DNA methylation
KW - Enzyme mechanism
KW - Site-directed mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=0038392721&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0038392721&partnerID=8YFLogxK
U2 - 10.1016/S0022-2836(03)00509-6
DO - 10.1016/S0022-2836(03)00509-6
M3 - Article
C2 - 12787669
AN - SCOPUS:0038392721
SN - 0022-2836
VL - 329
SP - 675
EP - 684
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -