Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Nucleic Acids Res. 2022 Dec 9;50(22):12896-12912. doi: 10.1093/nar/gkac1146.
The replicative DNA helicase translocates on single-stranded DNA to drive replication forks during chromosome replication. In most bacteria the ubiquitous replicative helicase, DnaB, co-evolved with the accessory subunit DciA, but how they function remains incompletely understood. Here, using the model bacterium Caulobacter crescentus, we demonstrate that DciA plays a prominent role in DNA replication fork maintenance. Cell cycle analyses using a synchronized Caulobacter cell population showed that cells devoid of DciA exhibit a severe delay in fork progression. Biochemical characterization revealed that the DnaB helicase in its default state forms a hexamer that inhibits self-loading onto single-stranded DNA. We found that upon binding to DciA, the DnaB hexamer undergoes conformational changes required for encircling single-stranded DNA, thereby establishing the replication fork. Further investigation of the functional structure of DciA revealed that the C-terminus of DciA includes conserved leucine residues responsible for DnaB binding and is essential for DciA in vivo functions. We propose that DciA stimulates loading of DnaB onto single strands through topological isomerization of the DnaB structure, thereby ensuring fork progression. Given that the DnaB-DciA modules are widespread among eubacterial species, our findings suggest that a common mechanism underlies chromosome replication.
复制 DNA 解旋酶在单链 DNA 上移动，以在染色体复制过程中驱动复制叉。在大多数细菌中，普遍存在的复制解旋酶 DnaB 与辅助亚基 DciA 共同进化，但它们的功能仍不完全清楚。在这里，我们使用模式细菌新月柄杆菌（Caulobacter crescentus）证明了 DciA 在 DNA 复制叉维持中发挥着重要作用。使用同步化的新月柄杆菌细胞群体进行的细胞周期分析表明，缺乏 DciA 的细胞在叉进展中表现出严重的延迟。生化特性表明，处于默认状态的 DnaB 解旋酶形成六聚体，抑制自身加载到单链 DNA 上。我们发现，与 DciA 结合后，DnaB 六聚体发生构象变化，需要环绕单链 DNA，从而建立复制叉。对 DciA 功能结构的进一步研究表明，DciA 的 C 末端包含保守的亮氨酸残基，负责 DnaB 结合，对于 DciA 的体内功能至关重要。我们提出，DciA 通过拓扑异构酶化 DnaB 结构来刺激 DnaB 加载到单链上，从而确保叉进展。鉴于 DnaB-DciA 模块在真细菌物种中广泛存在，我们的发现表明染色体复制存在共同的机制。
