DNA induces conformational changes in a recombinant human minichromosome maintenance complex.
DNA induces conformational changes in a recombinant human minichromosome maintenance complex.
复制标题
DOI：
10.1074/jbc.m114.622738
复制
发表时间：
2015-03-20
期刊：
影响因子：
--
通讯作者：
Chong JP
中科院分区：
文献类型：
--
作者：
Hesketh EL;Parker-Manuel RP;Chaban Y;Satti R;Coverley D;Orlova EV;Chong JP
背景：人类微小染色体维持复合体（hMCM）是DNA复制装置的重要组成部分。结果：hMCM在大肠杆菌中产生后，具有ATP酶和DNA解旋酶活性，并与DNA结合后发生构象变化。结论：重组hMCM在体外具有功能性。意义：hMCM为人类复制解旋酶的生化重建提供了重要工具。ATP依赖的DNA解旋活性已被证明为重组古细菌homohexameric minichromosome maintenance（MCM）复合物和它们的酵母heterohexameric对应物，但在高等真核生物，如果蝇，MCM相关的DNA解旋酶活性已被观察到的背景下，共纯化的Cdc 45-MCM-GINS复合物。在这里，我们描述了生产的重组人MCM（hMCM）复杂的大肠杆菌。这种蛋白质显示ATP水解活性，并且能够解旋双链体DNA。使用单粒子不对称EM重建，我们表明，重组hMCM形成一个六聚体，当绑定到DNA时，经历了构象变化。不经翻译后修饰而产生的重组hMCM在体外具有功能，并为人复制性解旋酶的生化重建提供了重要工具。
Background: The human minichromosome maintenance (hMCM) complex is an important component of the DNA replication apparatus. Results: After being produced in Escherichia coli, hMCM has ATPase and DNA helicase activity and undergoes a conformational change when bound to DNA. Conclusion: Recombinant hMCM is functional in vitro. Significance: hMCM provides an important tool for the biochemical reconstitution of the human replicative helicase. ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.