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.