Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Quartier UNIL/Sorge, Lausanne, CH 1015, Switzerland.
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Microbiology (Reading). 2013 Nov;159(Pt 11):2237-2248. doi: 10.1099/mic.0.068577-0. Epub 2013 Aug 23.
The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.
染色体复制的启动在细菌中受到严格调控，以确保每个细胞周期只发生一次。在许多变形菌中，这个过程需要 DnaA 蛋白的 ATP 结合形式。DnaA 的调节失活（RIDA）促进了 DnaA-ATP 向无复制活性的 DnaA-ADP 的转化，从而防止了过度起始。HdaA 蛋白的同源物与 DNA 聚合酶的 β-夹（DnaN）一起，是这个过程所必需的。在这里，我们使用荧光共振能量转移实验证明了 HdaA 在活的新月柄杆菌细胞中与 DnaN 相互作用。我们表明，HdaA 的 N 端区域中的 QFKLPL 基序是这种相互作用所必需的，并且该基序也需要将 HdaA 招募到 DNA 复制过程中复制体占据的亚细胞位置。不能与 DnaN 共定位或相互作用的 HdaA 突变蛋白也不能支持 HdaA 的基本功能。这些结果表明，在 RIDA 过程中，HdaA 被招募到复制体中，这可能是一种在 DnaA 失活之前感知染色体复制是否已经开始的方法。此外，我们还表明，HdaA 的 AAA+结构域中位于保守 R145 残基也需要 HdaA 的功能，尽管它不影响 HdaA 与 DnaN 在体内的相互作用。因此，HdaA 的 AAA+结构域可能在 DnaN 最初将 HdaA 招募到复制体之后的 RIDA 期间需要。
