Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.
Nucleic Acids Res. 2013 May;41(9):4860-76. doi: 10.1093/nar/gkt191. Epub 2013 Mar 27.
DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.
DNA 甲基转移酶 1（Dnmt1）在哺乳动物细胞中重新建立在 DNA 复制过程中产生的半甲基化 CpG 位点的甲基化。两个亚结构域，增殖细胞核抗原（PCNA）结合域（PBD）和靶向序列（TS）域，将 Dnmt1 靶向 S 期的复制位点。我们旨在剖析这两个结构域在细胞周期依赖性协调活动的细节。为此，我们结合了超分辨率 3D 结构照明显微镜和 GFP-Dnmt1 野生型和突变体构建体的荧光恢复后光漂白（FRAP）实验在体鼠细胞中。为了解释 FRAP 动力学的差异，我们改进了现有的数据分析和建模方法，以（i）解释在不同细胞周期阶段 Dnmt1 结合位点的不均匀和可变分布；（ii）允许扩散耦合动力学；（iii）适应多个结合类。我们发现，在早期 S 期（停留时间 Tres ≤ 10 s），瞬时 PBD 依赖性相互作用直接在复制位点是主要的特异性相互作用。在晚期 S 期，这种结合类被在富含 PCNA 的复制位点和附近着丝粒异染色质亚区的 TS 域依赖性相互作用所取代，该相互作用要强得多（Tres ∼22 s）。我们提出了一个双加载平台模型，用于在复制后异染色质 Dnmt1 靶位点进行额外的 PCNA 独立加载，以确保致密甲基化基因组区域的忠实维持。
