A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin
A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin
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DOI：
10.1074/jbc.m708684200
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发表时间：
2008-03-28
影响因子：
4.8
通讯作者：
Katayama, Tsutomu
中科院分区：
文献类型：
--
作者：
Ozaki, Shogo;Kawakami, Hironori;Katayama, Tsutomu
染色体复制的启动及其细胞周期的协调调节在大多数细胞生物中具有关键和基本的机制。大肠杆菌DnaA蛋白与复制起点（oriC）形成同源多聚体复合物。ATP-DnaA多聚体在oriC解旋元件（DUE）内解旋双链体。在这项研究中，结构分析表明，暴露在DnaA多聚体的推定结构的中心孔的几个残基可能是重要的解旋。使用突变分析，我们发现，在这些候选残基中，DnaA瓦尔-211和Arg-245是体内和体外起始的先决条件。而DnaA V211 A和R245 A蛋白保留正常的亲和力ATP/ADP和DNA和活性的ATP特异性构象变化的起始复合物在体外，oriC复合物的这些突变体蛋白是无活性的DUE解旋和结合的单链DUE。与包括ADP-DnaA或突变体DnaA的oriC复合物不同，ATP-DnaA-oriC复合物特异性结合单链DUE的上链。结合需要链内的特异性富T序列。在海洋栖热袍菌（Thermotoga maritima，一种古老的真细菌）中，DnaA直系同源物的相应保守残基也是DUE解旋所需的，这与DUE解旋的机制和调控可以在进化上保守的观点一致。这些发现提供了新的见解孔介导的起源解旋，ATP/ADP依赖性调节，和解旋酶加载的起始复合物的机制。
Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.