Mechanistic aspects of DnaA–RepA interaction as revealed by yeast forward and reverse two-hybrid analysis

Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein–protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system.

本研究借助酵母正反双杂交分析（yeast forward and reverse two-hybrid analysis）与生物化学技术（biochemical techniques），获取了全新且确凿的体内（in vivo）与体外（in vitro）实验证据，证实大肠杆菌（Escherichia coli）宿主编码的DnaA起始蛋白（DnaA initiator protein）的N末端结构域I与C末端结构域IV，均可与pSC101质粒（pSC101）编码的RepA起始蛋白（RepA initiator protein）发生物理相互作用。RepA的N端区域而非C端区域，可在体外与DnaA发生相互作用。此类蛋白质相互作用对于复制起始的两个关键早期步骤——即复制原点解旋与解旋酶加载——至关重要。单独的DnaA结构域I或结构域IV均无法与RepA协同以促进pSC101质粒的复制。然而，若将这两个结构域在同一细胞微环境中共表达，并通过一对亮氨酸拉链（leucine zipper）使其以非共价方式相互结合，则可在体内支持该质粒的复制。由此可见，将DnaA的结构域I与IV进行物理锚定（无论采用共价还是非共价方式），且两个结构域均能与RepA发生相互作用，是复制起始的关键条件。本研究结果还为一种基于复制机制的新型潜在细菌双杂交系统提供了分子层面的理论依据。