Caulobacter crescentus β sliding clamp employs a noncanonical regulatory model of DNA replication
Caulobacter crescentus β sliding clamp employs a noncanonical regulatory model of DNA replication
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Caulobactercrescentus β 滑动夹采用非规范的 DNA 复制调节模型。
DOI：
10.1111/febs.15138
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发表时间：
2019-11-29
期刊：
影响因子：
5.4
通讯作者：
Li, Xu
中科院分区：
文献类型：
--
作者：
Jiang, Xuguang;Zhang, Linjuan;Li, Xu
真细菌 β 滑动钳 (DnaN) 通过与 DNA、聚合酶和多种蛋白质因子直接相互作用，在 DNA 代谢中发挥着至关重要的作用。在大肠杆菌和其他一些物种中已经发现了典型的蛋白质-DnaN 相互作用，在此过程中，蛋白质伴侣通过共有的 β 结合基序被束缚在 DnaN 的保守典型疏水缝隙中。新月柄杆菌因其独特的不对称细胞分裂模式和有限的复制起始而成为研究 DNA 复制和细胞周期调控的优秀研究模型；然而，人们对 C. crescentus DnaN (CcDnaN) 的具体特征知之甚少。在这里，我们根据对接分析和晶体结构报告了 CcDnaN 与蛋白质的关联存在显着差异，表明其蛋白质伴侣的 β 结合基序结合了一个新的口袋而不是经典位点。 Pull-down 和等温滴定量热法结果表明，新口袋内的突变破坏了蛋白质-CcDnaN 相互作用。 DnaA 的复制和调节失活实验还表明，新口袋介导的蛋白质相互作用与复制过程中 CcDnaN 的性能和 DnaN 介导的调节过程密切相关。此外，对夹竞争的评估表明，DNA 在与新口袋结合时不会与蛋白质伴侣竞争。总体而言，我们的结构和生化分析提供了强有力的证据，证明 CcDnaN 采用非规范蛋白质关联模式。
The eubacterial beta sliding clamp (DnaN) plays a crucial role in DNA metabolism through direct interactions with DNA, polymerases, and a variety of protein factors. A canonical protein-DnaN interaction has been identified in Escherichia coli and some other species, during which protein partners are tethered into the conserved canonical hydrophobic crevice of DnaN via the consensus beta-binding motif. Caulobacter crescentus is an excellent research model for use in the investigation of DNA replication and cell-cycle regulation due to its unique asymmetric cell division pattern with restricted replication initiation; however, little is known about the specific features of C. crescentus DnaN (CcDnaN). Here, we report a significant divergence in the association of CcDnaN with proteins based on docking analysis and crystal structures that show that the beta-binding motifs of its protein partners bind a novel pocket instead of the canonical site. Pull-down and isothermal titration calorimetry results revealed that mutations within the novel pocket disrupt protein-CcDnaN interactions. It was also shown by replication and regulatory inactivation of DnaA assays that mediation of protein interaction by the novel pocket is closely related to the performance of CcDnaN during replication and the DnaN-mediated regulation process. Moreover, assessments of clamp competition showed that DNA does not compete with protein partners when binding to the novel pocket. Overall, our structural and biochemical analyses provide strong evidence that CcDnaN employs a noncanonical protein association pattern.