DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets
