Stenzel T T, MacAllister T, Bastia D
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Genes Dev. 1991 Aug;5(8):1453-63. doi: 10.1101/gad.5.8.1453.
We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101. We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw. This cooperative interaction at a distance, most probably, caused looping out of the ihf site. We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping. The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site. Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro. On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin.
我们研究了宿主编码的DNA弯曲蛋白IHF、宿主编码的起始蛋白DnaA以及质粒编码的起始蛋白RepA与pSC101复制起点的相互作用。我们发现，体外由IHF诱导的DNA弯曲促进了DnaA蛋白与两个物理上分离的结合位点dnaAs和dnaAw的相互作用。这种远距离的协同相互作用很可能导致ihf位点形成环出结构。我们还发现，RepA蛋白与其同源位点的结合促进了DnaA蛋白与物理上较远的dnaAs位点的增强结合，可能也是通过DNA环化实现的。将RepA添加到含有IHF和DnaA的结合反应中，进一步增强了DnaA蛋白与dnaAs位点的结合。因此，这三种DNA结合蛋白与复制起点相互作用，在体外产生了一种高阶结构。基于这三种蛋白对复制起始的已知需求结果，我们提出了一个复制起点预起始复合物结构的模型。
