Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis
Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis
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DOI：
10.1073/pnas.0506914103
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发表时间：
2006-02-14
影响因子：
11.1
通讯作者：
Noirot, P
中科院分区：
文献类型：
--
作者：
Noirot-Gros, MF;Velten, M;Noirot, P
DNA复制起始的调控对于确保基因组在每个细胞周期内只复制一次至关重要。在革兰氏阳性菌枯草芽孢杆菌中，YabA蛋白在起始控制中的功能是基于其与酵母双杂交中DnaA引发剂和DnaN滑动钳的相互作用以及在YabA无效菌株中观察到的过度起始表型。然而，YabA与已知的起始调节因子无关，并与一些其他蛋白质相互作用，这些蛋白质也可能直接或不参与起始控制。在这里，我们研究了YabA与DnaA和DnaN相互作用在起始控制中的具体作用，通过鉴定YabA中单个氨基酸的变化，该变化仅破坏了与DnaA或DnaN的相互作用。这些破坏性突变描述了YabA中涉及Zn2+簇结构的特定相互作用表面。在枯草芽孢杆菌中，这些YabA相互作用突变取消了起始控制和YabA在复制工厂的形成。当缺失的YabA突变体共表达时，混合低聚物在复制体上形成焦点，并恢复了起始控制，表明YabA与dna和DnaN在异复合体中起作用。与此一致的是，纯化的YabA寡聚并与dna和DnaN形成配合物。这些发现强调了YabA与复制机制的功能关联，表明YabA通过与复制延伸的偶联来调节起始。
The regulation of initiation of DNA replication is crucial to ensure that the genome is replicated only once per cell cycle. In the Gram-positive bacterium Bacillus subtilis, the function of the YabA protein in initiation control was assigned based on its interaction with the DnaA initiator and the DnaN sliding clamp in the yeast two-hybrid and on the overinitiation phenotype observed in a yabA null strain. However, YabA is unrelated to known regulators of initiation and interacts with several additional proteins that could also be involved directly or not in initiation control. Here, we investigated the specific role of YabA interactions with DnaA and DnaN in initiation control by identifying single amino acid changes in YabA that disrupted solely the interaction with DnaA or DnaN. These disruptive mutations delineated specific interacting surfaces involving a Zn2+-cluster structure in YabA. In B. subtilis, these YabA interaction mutations abolished both initiation control and the formation of YabA foci at the replication factory. Upon coexpression of deficient YabA mutants, mixed oligomers formed foci at the replisome and restored initiation control, indicating that YabA acts within a heterocomplex with DnaA and DnaN. In agreement, purified YabA oligomerized and formed complexes with DnaA and DnaN. These findings underscore the functional association of YabA with the replication machinery, indicating that YabA regulates initiation through coupling with the elongation of replication.