Mcm Subunits Can Assemble into Two Different Active Unwinding Complexes
Mcm Subunits Can Assemble into Two Different Active Unwinding Complexes
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DOI：
10.1074/jbc.m804686200
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发表时间：
2008-11-07
影响因子：
4.8
通讯作者：
Kaplan, Daniel L.
中科院分区：
文献类型：
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作者：
Kanter, Diane M.;Bruck, Irina;Kaplan, Daniel L.
真核生物中的复制叉解旋酶是由Mcm 2 -7、Cdc 45和GINS组成的大型复合物。Mcm 2 -7蛋白形成一个异六聚体环，水解ATP并为这种解旋复合物提供运动功能。一个全面的研究如何个别Mcm亚基生化活动与解旋功能尚未完成。我们研究了Mcm 4-Mcm 6-Mcm 7复合物的机制，该复合物是一个有用的模型系统，因为该复合物在体外具有解旋酶活性。我们分别纯化了三个Mcm亚基中的每一个，直到它们都不含核酸酶，然后我们研究了Mcm亚基不同组合的生化特性。我们发现Mcm 4和Mcm 7形成了一个活跃的解旋组装体。将Mcm 6添加到Mcm 4/Mcm 7导致形成活性Mcm 4/Mcm 6/Mcm 7解旋酶组装体。Mcm 4-Mcm 7复合物形成环状六聚体，其通过空间排阻机制以3'至5'极性解旋DNA，类似于Mcm 4/Mcm 6/Mcm 7。Mcm 4-Mcm 7复合物具有高水平的ATP酶活性，其进一步被DNA刺激。不同的Mcm混合物形成环或表现出ATP酶活性的DNA刺激的能力与这些复合物解开DNA的能力相关。Mcm 4/Mcm 7和Mcm 4/Mcm 6/Mcm 7组装体可以打开以加载到环状DNA上以启动解旋。我们的结论是，Mcm亚基是令人惊讶的灵活性和动态的能力，相互作用，形成积极的解旋复合物。
The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.