Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

哺乳动物 DNA 甲基化模式由两种从头 DNA 甲基转移酶 DNMT3A 和 DNMT3B 建立，它们表现出冗余和独特的甲基化活性。然而，相关的分子基础仍未确定。通过对 DNMT3A 和 DNMT3B 的全面结构、酶学和细胞表征，我们在此报告了支持其不同基因组甲基化活性的多层底物识别机制。DNMT3B 催化环中的氢键导致 CpG 特异性低于 DNMT3A，而靶标识别域和同源二聚体界面的相互作用微调了两种酶之间的不同靶标选择，DNMT3B 的赖氨酸 777 充当独特的传感器+1 侧翼基地。DNMT3B突变。总之，这项研究揭示了两种 DNMT3 酶的独特底物读出机制，暗示它们在发育和发病机制中的不同作用。