The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.In mammals, the methylation of cytosines represents the only known form of covalent DNA modification that has a clear biological function, through its association with stable transcriptional silencing (1). Such silencing is critical for proper embryonic development, genome stability, X chromosome inactivation, genomic imprinting, and the silencing of retrotransposons (2-4). In addition, aberrant DNA methylation underlies many human diseases, including cancer, which is often associated with a genome-wide loss of DNA methylation and inappropriate silencing of tumor suppressor genes (5, 6). DNA methyltransferases (DNMTs) 2 are essential enzymes that catalyze the transfer of a methyl group from a donor molecule, S-adenosyl-L-methionine (SAM), to a cytosine ring, usually located within the context of a symmetrical CpG dinucleotide (7). Mammalian genomes carry three distinct active DNMT genes. Two de novo DNMTs, encoded by the DNMT3A and DNMT3B genes, have been identified (8). These proteins show a high similarity to each other and have several domains in common, including a highly conserved C-terminal domain containing catalytic DNMT motifs that are homologous to those originally defined for bacterial type II cytosine DNMTs (9). DNMT3A and DNMT3B are considered to be de novo DNMTs, since they are responsible for initiating DNA methylation during early embryonic development (10), and show a preference for unmethylated DNA in vitro (8,11). Specif...
