title: Targeted mutagenesis results in an activation of DNA methyltransferase 1 and confirms an autoinhibitory role of its RFTS domain.
abstract: The N-terminal regulatory part of DNA methyltransferase 1 (Dnmt1) contains a replication foci targeting sequence (RFTS) domain, which is involved in the recruitment of Dnmt1 to replication forks. The RFTS domain has been observed in a crystal structure to bind to the catalytic domain of the enzyme and block its catalytic centre. Removal of the RFTS domain led to activation of Dnmt1, thus suggesting an autoinhibitory role of this domain. Here, we destabilised the interaction of the RFTS domain with the catalytic domain by site-directed mutagenesis and purified the corresponding Dnmt1 variants. Our data show that these mutations resulted in an up to fourfold increase in Dnmt1 methylation activity in vitro. Activation of Dnmt1 was not accompanied by a change in its preference for methylation of hemimethylated CpG sites. We also show that the Dnmt1 E572R/D575R variant has a higher DNA methylation activity in human cells after transfection into HCT116 cells, which are hypomorphic for Dnmt1. Our findings strongly support the autoinhibitory role of the RFTS domain, and indicate that it contributes to the regulation of Dnmt1 activity in cells.
authors: Institute of Biochemistry, Faculty of Chemistry, University Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart (Germany).
authors: Pavel
authors: P
authors: Bashtrykov
authors: Arumugam
authors: A
authors: Rajavelu
authors: Benjamin
authors: B
authors: Hackner
authors: Sergey
authors: S
authors: Ragozin
authors: Thomas
authors: T
authors: Carell
authors: Albert
authors: A
authors: Jeltsch
mesh_terms: D020134:Catalytic Domain; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D004789:Enzyme Activation; D006801:Humans; D008958:Models, Molecular; D017354:Point Mutation; D013379:Substrate Specificity
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human
keywords: DNA methylation; DNA methyltransferases; enzyme catalysis; enzyme specificity