Mechanisms for ATP-dependent activation of highly-ordered complex formed by the initiator protein DnaA.

ATP-DnaA在复制起点oriC的DnaA组装区（DAR）上多聚化，使DAR侧翼的双链体解旋元件（DUE）解旋。ATP-DnaA-oriC复合物结合所得单链（ss）DUE，促进DnaB解旋酶加载。然而，DUE解旋的机制仍不清楚。在这里，使用各种体外重建系统，我们确定功能不同的DnaA亚复合物上DAR形成，并揭示其特定的作用和新的机制，在DUE解旋。DUE侧翼的左半部分DAR携带高亲和力的DnaA盒R1和几个ATP-DnaA优先位点，形成了一个DnaA亚复合体，能够进行DUE解旋和ssDUE结合，从而支持基础DnaB负载活性。该亚复合物进一步细分为两个;在ATP-DnaA-优先位点上形成的DUE-远端DnaA亚复合物结合ssDUE。在DnaA盒R1上形成的DUE侧翼DnaA亚复合物与DNA弯曲蛋白IHF合作将DUE招募到DUE远端DnaA亚复合物，促进起始。这些动力学在初始复合物中可能是真细菌进化中保守的。

ATP-DnaA multimerize on the DnaA-assembly region (DAR) of the replication origin oriC, unwinding the duplex unwinding element (DUE) flanking DAR. The ATP-DnaA-oriC complex binds the resultant single-stranded (ss)DUE, promoting DnaB helicase loading. However, mechanisms in DUE unwinding remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal their specific roles and novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and several ATP-DnaA-preferential sites formed a DnaA sub-complex competent to DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided to two ; The DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. The DUE-flanking DnaA sub-complex formed on DnaA box R1 recruits DUE to the DUE-distal DnaA sub-complex cooperatively with a DNA-bending protein IHF, promoting initiation. These dynamics in the initial complexes are likely conserved in eubacterial evolution.