Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA-and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.DNA cytosine methylation, an epigenetic mark that occurs predominantly at CpG dinucleotides, is the principal eukaryotic DNA modification (1). DNA methylation is a key component of gene silencing, which makes significant contributions to cell phenotype. Establishment and maintenance of DNA methylation patterns are governed by three catalytically active DNA methyltransferases: Dnmt3a, Dnmt3b, and Dnmt1. The Dnmt3 isoforms are responsible for de novo methylation during germ cell and embryonic development and bind with high affinity to unmethylated DNA sequences (2). CpG methylation patterns are maintained in mammals by Dnmt1 with hemimethylated CpG dinucleotides serving as preferred substrates.Human Dnmt1 (1616 amino acids) consists of a conserved C-terminal catalytic core (amino acids 1140 -1616) and a large N-terminal region (amino acids 1-1139) harboring multiple globular conserved domains, including the DMAP1 (DNA methyltransferase-associated protein 1)-binding domain (3), the proliferating cell nuclear antigen-binding domain (4), the replication focus targeting sequence (RFTS) 4 domain (residues 351-600) (5), the CXXC domain (6), and two bromo-adjacent homology (BAH) domains (see Fig. 1 ) (7).The CXXC domain is understood to contribute to catalytic activity by interacting with unmethylated CpG DNA substrates (6,8). This was observed in the recently solved crystal structures of Dnmt1, which encompass sequences from the CXXC domain to the C terminus (9). In the structures, the CXXC domain binds and holds unmethylated duplex CpG-containing DNA away from the active site, whereas the acidic linker between the CXXC and BAH1 domains is bound in the active site between the DNA segment and the S-adenosylhomocysteine product (9). This observation helps to explain the relationship between the CXXC and catalytic domains. However, it does not address the role of domains N-terminal to the CXXC domain, which are not present in the structures.We set out to clarify the structure and function of the RFTS domain. The RFTS domain is conserved by sequence (supplemental Fig. S1) and cont...