Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Institute for Systems Theory and Automatic Control, University of Stuttgart, Pfaffenwaldring 9, 70569 Stuttgart, Germany.
Nucleic Acids Res. 2023 Jul 21;51(13):6622-6633. doi: 10.1093/nar/gkad465.
The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.
DNMT1 对半甲基化 DNA 的特异性是 DNA 甲基化遗传的核心特征。我们使用具有单个 CpG 位点的随机序列背景中的半甲基化 (HM)、半羟甲基化 (OH) 和未甲基化 (UM) 底物，在竞争甲基化动力学中研究了这一特性。DNMT1 对半甲基化/未甲基化底物的侧翼序列依赖性 HM/UM 特异性平均为 80 倍，在长半甲基化 DNA 底物上略有增强。为了解释单个甲基基团的强烈影响，我们提出了一个新模型，其中 5mC 甲基基团的存在通过空间排斥将 DNMT1-DNA 复合物的构象改变为活性构象。HM/OH 偏好与侧翼序列有关，平均只有 13 倍，表明在许多侧翼环境中，5hmC 生成的被动 DNA 去甲基化效率不高。在 DNA 与 DNMT1 结合期间，DNMT1 的 CXXC 结构域对半甲基化/未甲基化特异性具有适度的侧翼序列依赖性贡献，但如果 DNMT1 以连续甲基化模式甲基化长 DNA 分子，则没有贡献。将来自具有各种 DNMT 和 TET 缺失的小鼠 ES 细胞系的基因组甲基化模式与我们的数据进行比较，发现 UM 特异性谱与细胞甲基化模式最相关，表明 DNMT1 的从头甲基化活性在这些细胞中塑造了 DNA 甲基组。
