Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Department of Biochemistry, University of California, Riverside, CA 92521, USA.
Nucleic Acids Res. 2020 Nov 18;48(20):11495-11509. doi: 10.1093/nar/gkaa938.
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.
DNA 甲基转移酶在可变侧翼序列的背景下与它们的 CpG 靶位相互作用。我们使用含有随机侧翼序列中 CpX 靶位的底物池研究了人类 DNMT3B 催化结构域的 DNA 甲基化，并确定了 CpG 识别和侧翼序列相互作用以及涉及平衡与不同侧翼位点相互作用的复杂接触网络的综合效应。侧翼序列对非 CpG 位点的 DNA 甲基化率的影响大于对 CpG 位点的影响。我们表明 T775 在 DNMT3B 的催化机制中具有重要的动态作用。此外，我们在 DNMT3B 的 DNA 结合界面中鉴定了六个氨基酸残基（N652、N656、N658、K777、N779 和 R823），这些残基通过与侧翼残基形成补偿相互作用，参与平衡有利和不利序列环境中 CpG 位点的甲基化率，包括与+1 侧翼位点的 A 形成 CpG 特异性接触。DNMT3B 的非 CpG 侧翼偏好与人类细胞中非 CpG 甲基化模式高度相关。人与小鼠 DNMT3B 侧翼序列偏好的比较显示出细微的差异，表明侧翼序列偏好和细胞 DNMT 靶标共同进化。
