Zhu B, Zheng Y, Angliker H, Schwarz S, Thiry S, Siegmann M, Jost J P
Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Nucleic Acids Res. 2000 Nov 1;28(21):4157-65. doi: 10.1093/nar/28.21.4157.
A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.
分离并测序了一段1468 bp的cDNA，其编码人类MBD4 G/T错配DNA糖基化酶的鸡同源物。推导的氨基酸序列（416个氨基酸）与人类MBD4有46%的同一性，且C末端的保守催化区域（170个氨基酸）有90%的同一性。禽类蛋白的非保守区域没有甲基化DNA结合结构域的共有序列。来自人和鸡的重组蛋白具有G/T错配以及5-甲基胞嘧啶（5-MeC）DNA糖基化酶活性。通过凝胶迁移试验检测时，有或没有甲基化DNA结合结构域的人类重组蛋白与对称的、半甲基化DNA和非甲基化DNA的结合能力相同。然而，该酶对半甲基化DNA仅具有5-MeC DNA糖基化酶活性。对人类MBD4和一个N末端缺失突变体与部分脱嘌呤和脱嘧啶底物进行足迹分析，揭示了这些蛋白对CpG周围修饰底物的选择性结合。至于从鸡胚中纯化的5-MeC DNA糖基化酶，MBD4不使用含有mCpA、mCpT或mCpC的寡核苷酸作为底物。富含A+T的寡核苷酸中的mCpG比A+T含量低的序列是更好的底物。人类MBD4对半甲基化DNA的K:(m)约为10(-7) M，V:(max)约为10(-11) mol/h/μg蛋白。缺失突变表明G/T错配和5-MeC DNA糖基化酶位于C末端保守区域。与从鸡胚DNA去甲基化复合物中分离的5-MeC DNA糖基化酶形成鲜明对比的是，MBD4的两种酶活性受到RNA的强烈抑制。用反义RNA进行原位杂交表明，MBD4仅位于分化胚胎组织的分裂细胞中。
