Funnell B E
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Aug 5;266(22):14328-37.
The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.
P1 ParB蛋白是质粒原噬菌体进行活性分配从而实现稳定遗传所必需的。ParB和大肠杆菌蛋白整合宿主因子（IHF）参与在类着丝粒位点parS处组装分配复合物。在本报告中，对IHF在分配复合物形成中的作用进行了探索。首先，为这些研究纯化了ParB蛋白，结果表明ParB在溶液中形成二聚体。接下来，将IHF结合位点定位到parS内一个29个碱基对的区域，包括序列TAACTGACTGTTT（在两个位置上与IHF共有序列不同）。IHF在其结合位点处诱导DNA发生强烈弯曲。在体外和体内，失去或破坏了IHF结合位点的parS版本与ParB的结合亲和力大大降低。结合常数的测量表明，IHF使ParB对野生型parS位点的亲和力提高了约10000倍。最后，DNA超螺旋在有IHF存在时能改善ParB的结合，但在没有IHF时则不然。这些观察结果表明，IHF和超螺旋通过促进ParB在parS处的精确定位来协助ParB，这种空间排列导致ParB对parS具有高亲和力。
