DNA replication initiation in Bacillus subtilis ; Structural and functional characterisation of the essential DnaA-DnaD interaction
DNA replication initiation in Bacillus subtilis ; Structural and functional characterisation of the essential DnaA-DnaD interaction
复制标题
枯草芽孢杆菌中 DNA 复制起始；
DOI：
10.1101/444885
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Martin E
中科院分区：
文献类型：
--
作者：
Martin E
同四聚体 DnaD 蛋白在低 G+C 含量的革兰氏阳性细菌中是必需的，并且参与复制起始和折叠复制叉的重新启动。它在理论上与普遍保守的细菌主复制起始蛋白 DnaA 相互作用，但缺乏这种相互作用的结构和功能细节，从而导致我们对支持细菌复制起始的分子细节的不完全理解。 DnaD 包含 N 端 (DDBH1) 和 C 端 (DDBH2) 结构域，与细菌双杂交和酵母双杂交研究相矛盾，表明前者或后者分别与 DnaA 相互作用。使用核磁共振 (NMR)，我们表明 DDBH1 和 DDBH2 均与 DnaA 的 N 端结构域 I 相互作用，并研究了 DDBH2 相互作用的结构细节。我们揭示了 DDBH2-DnaA 结构域 I 复合物的两个构象家族，并表明 DnaD 的 DnaA 相互作用斑块与 DNA 相互作用斑块不同，这表明 DnaD 可以同时结合 DNA 和 DnaA。使用敏感的单分子 FRET 技术，我们发现 DnaD 重塑 DnaA-DNA 丝，与拉伸和/或解扭一致。此外，DnaD 的 DNA 结合活性对于这种细丝重塑来说是多余的。这反过来表明，DnaA 和 DnaD 在理论上协同工作，在复制起始过程中局部熔化 DNA 双链体。
The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation atoriCand re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at theoriCbut structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA–DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in theoriCto locally melt the DNA duplex during replication initiation.