Datta H J, Khatri G S, Bastia D
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):73-8. doi: 10.1073/pnas.96.1.73.
Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA-DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA-DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.
尽管许多细菌染色体仅需一种复制起始蛋白，例如DnaA，但大多数质粒复制子依赖双重起始蛋白：宿主编码的DnaA和质粒编码的Rep起始蛋白来启动复制。本研究以质粒pSC101作为模型系统，探究了对双重起始蛋白需求的生物学原理，并表明质粒编码的RepA与复制解旋酶DnaB特异性相互作用。DnaB或RepA中破坏RepA-DnaB相互作用的突变会导致体外无法将DnaB加载到质粒ori上，以及体内无法复制质粒。尽管DnaA与DnaB的相互作用不能替代RepA-DnaB相互作用来进行解旋酶加载，但DnaA与整合宿主因子、DnaC和RepA一起对于解旋酶加载至关重要。因此，解旋酶加载间接需要DnaA。有趣的是，DnaB解旋酶似乎没有与起始蛋白相互作用的共同表面，而是至少有数量有限的不重叠表面，每个表面都与不同的起始蛋白特异性相互作用。
