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

引发剂滴定机制在染色体复制起始中的作用

基本信息

批准号：
13680761
负责人：
OGAWA Tohru
金额：
$ 1.92万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680761/
关键词：
DNA replication DnaA oriC initiation of replication datA IHF

项目摘要

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.

大肠杆菌染色体的复制起始于一个独特的位点oriC。同时启动发生在所有oriC网站目前在一个细胞一次，只有一次在一个固定的时间在细胞周期。确保循环起始事件通过染色体位点datA操作的机制，该染色体位点使异常大量的细菌起始蛋白DnaA发生突变，缺乏datA的菌株正常生长，但由于额外的起始事件而表现出异步起始表型。DnaA滴定机制，以控制复制起始似乎是，内在的datA，因为删除其他DnaA结合位点（DnaA盒）的染色体上不会引起这样的异常initiations.In本研究中，努力澄清的独特机制的DnaA滴定datA。在datA中的不同位点携带突变的菌株的表型分析显示，在datA中的五个DnaA框中，第二个和第三个框对于起始控制是必不可少的。整合宿主因子（IHF）结合的共有序列存在于两个DnaA盒之间。我们已经证明IHF蛋白确实与该位点结合。此外，IHF结合大大增强了滴定至datA的DnaA分子的量。已知IHF与其识别位点的结合导致DNA弯曲约160度。这种弯曲应使第二和第三个DnaA盒紧密接近，并使桥接的DNA链的DnaA-DnaA相互作用绑定到两个DnaA盒，导致形成一个大的DnaA滴定pool.Destruction的IHF结合位点的染色体上的datA引起overinitiation表型类似的datA-删除的菌株观察到的，证明了在体内DnaA滴定datA IHF的参与。