title: CXXC domain of human DNMT1 is essential for enzymatic activity.
abstract: DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 (DeltaCXXC)) localized on replication foci. Both point mutant and DNMT1 (DeltaCXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 (DeltaCXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938-2723, USA. pradhan@neb.com
authors.forename: Mihika
authors.initials: M
authors.lastname: Pradhan
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Estève
authors.forename: Hang Gyeong
authors.initials: HG
authors.lastname: Chin
authors.forename: Mala
authors.initials: M
authors.lastname: Samaranayke
authors.forename: Gun-Do
authors.initials: GD
authors.lastname: Kim
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan