You Zhiying, Ishimi Yukio, Masai Hisao, Hanaoka Fumio
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan.
J Biol Chem. 2002 Nov 8;277(45):42471-9. doi: 10.1074/jbc.M205769200. Epub 2002 Aug 30.
Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.
Mcm由六个结构相关的亚基（Mcm2 - 7）组成，对真核生物DNA复制至关重要。Mcm的一个亚组件，即Mcm4/6/7双三聚体复合物，具有DNA解旋酶活性，有人提出Mcm可能在复制叉处作为复制性解旋酶发挥作用。我们在此表明，Mcm7保守的ATP酶基序对于Mcm4/6/7复合物的ATP酶和DNA解旋酶活性至关重要。由于未复合的Mcm7既不显示ATP酶活性也不显示DNA解旋酶活性，Mcm7通过与其他亚基相互作用对Mcm复合物的DNA解旋酶活性有贡献。相反，含有DNA结合活性部分受损的Mcm4锌指突变体的Mcm4/6/7复合物表现出升高的DNA解旋酶活性。含有这种Mcm4突变体的Mcm4/6/7复合物倾向于解离为三聚体复合物，这表明Mcm4的锌指参与三聚体的亚基相互作用。缺少N端35或112个氨基酸的Mcm4突变体可以形成六聚体Mcm4/6/7复合物，但显示出非常低的DNA解旋酶活性。结合先前报道的Mcm6在ATP结合中的重要作用（You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003 - 8015），我们的数据表明Mcm4、Mcm6和Mcm7亚基在激活Mcm4/6/7复合物的DNA解旋酶活性中具有不同作用。
