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

ATP-Dna A在复制起点ORIC的Dna-组装区域(DAR)上多聚，解开DAR两侧的双链解离元件(DUE)。ATP-DNAA-ORIC复合体与生成的单链(Ss)结合，促进DNAB解旋酶的负载。然而，适当平仓的机制仍不清楚。在这里，我们使用不同的体外重组系统，鉴定在DAR上形成的不同功能的DNAA亚复合体，并揭示它们在适当解离中的特定作用和新的机制。适当侧翼的左半部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，具有适当的解离和单链DUE结合的能力，从而支持基本的DNAB负载活性。这个亚复合体进一步分为两个；在ATP-Dna A优先位点上形成的应有的远端Dna A亚复合体与ssDUE结合。在Dna A盒R1上形成的适当侧翼的Dna A亚复合体由于其末端的Dna A亚复合体与DNA弯曲蛋白IHF协同作用而招募新兵，促进了起始。初始复合体中的这些动力学很可能在真细菌进化中是保守的。

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.