Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.
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.
Gowher
Humaira
H
International University Bremen, Biochemistry, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany.
Loutchanwoot
Panida
P
Vorobjeva
Olga
O
Handa
Vikas
V
Jurkowska
Renata Z
RZ
Jurkowski
Tomasz P
TP
Jeltsch
Albert
A
D024509:3' Flanking Region / Q000235:genetics; D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D004252:DNA Mutational Analysis; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012436:S-Adenosylmethionine / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein
C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; D012436:S-Adenosylmethionine; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A