Manen D, Xia G, Caro L
Department of Molecular Biology, University of Geneva, Switzerland.
Mol Microbiol. 1994 Mar;11(5):875-84. doi: 10.1111/j.1365-2958.1994.tb00366.x.
The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds IR1 and the RepA monomers that bind the RS sequences.
质粒pSC101的复制起点包含三个直接重复序列RS1、RS2和RS3，它们与两个回文序列IR1和IR2相隔22个碱基对，这两个回文序列与直接重复序列部分同源。这些反向重复（IR）序列与repA基因的启动子重叠，repA基因编码质粒复制所必需的一种蛋白质。我们已经表明，RepA以单体形式结合到RS位点，以二聚体形式结合到IR位点。我们详细研究了IR1位点以及将其与RS3分隔开的DNA片段对质粒拷贝数控制的影响。我们发现，IR1的完整性对于高效复制和质粒稳定性至关重要，关键位点延伸到IR1本身的左侧。我们还发现，IR1的存在深刻改变了纯化的RepA蛋白与包含RS序列的DNA片段的结合特性。IR1与其在RS3上的同源位点被大约四圈DNA螺旋分隔开。如果这个距离增加半圈螺旋，复制就会被消除，但如果距离增加一整圈螺旋，复制就会恢复。这些结果表明，在复制起始过程中，结合IR1的RepA二聚体与结合RS序列的RepA单体之间存在DNA环化相互作用。
