From the Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2014 Jan 3;289(1):379-86. doi: 10.1074/jbc.M113.523209. Epub 2013 Nov 19.
Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.
Dnmt1 负责在复制过程中维持 DNA 甲基化，以便将甲基化模式传递给下一代。靶向复制焦点的序列（RFTS），插入催化口袋，对于招募 Dnmt1 到复制位点是必要的。在本研究中，我们发现 Dnmt1 的 DNA 甲基化活性与 DNA 长度有关，并且几乎不会甲基化 12 个碱基的短半甲基化 DNA。相反，RFTS 缺失的 Dnmt1 和破坏 RFTS 与催化结构域之间氢键的 Dnmt1 突变体即使在 12 个碱基的半甲基化 DNA 上也表现出显著的 DNA 甲基化活性。RFTS 缺失的 Dnmt1 对 12 个碱基的半甲基化 DNA 的 DNA 甲基化活性在添加 RFTS 时被强烈抑制，但对破坏氢键形成的 Dnmt1 抑制作用较小。Uhrf1 的 SRA 结构域是维持甲基化的必要因素，并且选择性地结合半甲基化 DNA，刺激 Dnmt1 的 DNA 甲基化活性。SRA 与 Dnmt1 的浓度比是最大刺激的决定因素。此外，一个失去 DNA 结合活性但仍能与 Dnmt1 结合的突变 SRA 刺激 Dnmt1 的 DNA 甲基化活性。结果表明，SRA 与 Dnmt1 的直接相互作用刺激了 Dnmt1 的 DNA 甲基化活性。SRA 促进了催化中心对半甲基化 12 个碱基氟胞嘧啶 DNA 的接受。我们提出，SRA 从催化口袋中移除 RFTS 塞，以促进催化中心对 DNA 的接受。
