Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading.
The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides. Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity. Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1. Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501. Peptides from the N- and C-termini bound methylated DNA independently. Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation. Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A). Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.
Pradhan
Sriharsa
S
New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA. pradhan@neb.com
Estève
Pierre-Olivier
PO
D000494:Allosteric Regulation; D000495:Allosteric Site; D020134:Catalytic Domain; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D017384:Sequence Deletion; D013379:Substrate Specificity; D014162:Transfection
D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D003596:Cytosine; D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D005982:Glutathione Transferase