Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.
Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.
J Mol Biol. 2021 Sep 17;433(19):167186. doi: 10.1016/j.jmb.2021.167186. Epub 2021 Aug 8.
DNA interacting enzymes recognize their target sequences embedded in variable flanking sequence context. The influence of flanking sequences on enzymatic activities of DNA methyltransferases (DNMTs) can be systematically studied with "deep enzymology" approaches using pools of double-stranded DNA substrates, which contain target sites in random flanking sequence context. After incubation with DNMTs and bisulfite conversion, the methylation states and flanking sequences of individual DNA molecules are determined by NGS. Deep enzymology studies with different human and mouse DNMTs revealed strong influences of flanking sequences on their CpG and non-CpG methylation activity and the structures of DNMT-DNA complexes. Differences in flanking sequence preferences of DNMT3A and DNMT3B were shown to be related to the prominent role of DNMT3B in the methylation of human SATII repeat elements. Mutational studies in DNMT3B discovered alternative interaction networks between the enzyme and the DNA leading to a partial equalization of the effects of different flanking sequences. Structural studies in DNMT1 revealed striking correlations between enzymatic activities and flanking sequence dependent conformational changes upon DNA binding. Correlation of the biochemical data with cellular methylation patterns demonstrated that flanking sequence preferences are an important parameter that influences genomic DNA methylation patterns together with other mechanisms targeting DNMTs to genomic sites.
DNA 相互作用酶识别其嵌入在可变侧翼序列背景中的靶序列。通过使用双链 DNA 底物的池，采用“深度酶学”方法可以系统地研究侧翼序列对 DNA 甲基转移酶 (DNMTs) 的酶活性的影响，这些底物在随机侧翼序列背景中包含靶位点。用 DNMTs 和亚硫酸氢盐转化孵育后，通过 NGS 确定单个 DNA 分子的甲基化状态和侧翼序列。用不同的人和小鼠 DNMT 进行的深度酶学研究表明，侧翼序列对其 CpG 和非 CpG 甲基化活性以及 DNMT-DNA 复合物的结构有很强的影响。DNMT3A 和 DNMT3B 的侧翼序列偏好差异与 DNMT3B 在人类 SATII 重复元件的甲基化中起着重要作用有关。在 DNMT3B 中的突变研究发现，酶与 DNA 之间存在替代的相互作用网络，导致不同侧翼序列的影响部分均等化。在 DNMT1 中的结构研究揭示了酶活性与 DNA 结合时侧翼序列依赖性构象变化之间的惊人相关性。生化数据与细胞甲基化模式的相关性表明，侧翼序列偏好是影响基因组 DNA 甲基化模式的一个重要参数，与将 DNMT 靶向基因组位点的其他机制一起。
