The DNMT1 target recognition domain resides in the N terminus.
DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.
Araujo
F D
FD
Departments of Pharmacology and Therapeutics, Biochemistry, and Experimental Medicine, McGill University, Montreal, PQ, H3G 1Y6, Canada.
Croteau
S
S
Slack
A D
AD
Milutinovic
S
S
Bigey
P
P
Price
G B
GB
Zannis-Hadjopoulos
M
M
Zannis-Hajopoulos
M
M
Szyf
M
M
D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry*; D017353:Gene Deletion; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D009841:Oligonucleotides; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human