Role of the initiator titration mechanism in the initiation of chromosomal replication

大肠埃希氏菌染色体的复制是在一个独特的位点ORIC开始的。细胞中的所有ORIC位点同时启动一次，并且在细胞周期中的固定时间只发生一次。一种确保循环启动事件通过染色体位置DATA进行操作的机制，它滴定异常大量的细菌启动蛋白DNAA，缺乏数据的菌株正常生长，但由于额外的启动事件而表现出异步启动表型。Dna A滴定控制复制启动的机制似乎是数据固有的，因为染色体上其他Dna A结合位点(Dna A盒)的缺失不会引起这种异常启动。在本研究中，试图通过数据来阐明Dna A滴定的独特机制。对在数据中不同位置携带突变的菌株的表型分析表明，在数据中的五个Dna A盒中，第二和第三个盒都是启动控制所必需的。在两个DNAA盒之间存在整合宿主因子(IHF)的共同结合序列。我们已经证明IHF蛋白确实能与这个位点结合。此外，IHF结合大大增加了滴定到数据的DNAA分子的数量。已知IHF与其识别位点的结合会导致DNA弯曲约160度。这种弯曲应该使第二个和第三个Dna A盒靠近，并通过与两个Dna A盒结合的Dna A-Dna A相互作用将DNA链连接起来，导致形成一个大的Dna A滴定池。染色体上数据中IHF结合位点的破坏导致了与数据缺失菌株相似的过度引发表型，表明IHF参与了体内数据的Dna A滴定。

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiations occur at all oriC sites present in a cell once and only once at a fixed time in the cell cycle. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, A strain lacking datA grew normally but exhibited the asynchronous initiation phenotype due to extra initiation events. The DnaA titration mechanism to control replication initiation appears to be, intrinsic to datA, inasmuch as deletion of other DnaA-binding sites (DnaA boxes) on the chromosome does not provoke such aberrant initiations.In the present study, efforts were made to clarify the unique mechanism of DnaA titration by datA. Analyses of phenotypes of strains carrying mutations at various sites in datA revealed that, of the five DnaA boxes in datA, both the second and the third boxes are essential for the initiation control. A consensus sequence of binding of the integration host factor (IHF) is present between the two DnaA boxes. We have proven that IHF protein really binds to this site. Furthermore, the IHF binding greatly enhanced the amounts of DnaA molecules titrated to datA. Binding of IHF to its recognition site has been known to cause DNA bending of about 160 degree. This bending should bring the second and the third DnaA boxes to close proximity and enable bridging the DNA strands by DnaA-DnaA interaction bound to the two DnaA boxes, leading to the formation of a large DnaA titration pool.Destruction of the IHF-binding site in datA on the chromosome caused overinitiation phenotype similar to that observed with the datA-deleted strain, demonstrating the involvement of IHF in DnaA titration to datA in vivo.