McEachern M J, Bott M A, Tooker P A, Helinski D R
Department of Biology, University of California at San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942-6. doi: 10.1073/pnas.86.20.7942.
The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.
复制起始因子π蛋白的γ原点结合位点由七个22碱基对（bp）的直接重复序列组成，先前已证明其对质粒R6K的复制至关重要，如果以反式提供，它在大肠杆菌细胞中也可作为R6K复制的抑制剂。抑制作用取决于这些重复序列结合R6K编码的π蛋白的能力，但不会因增加细胞内π水平而被克服。在γ原点附近插入第二个重复序列簇也可显著抑制复制。这种效应的严重程度取决于额外簇中重复序列的位置、方向和数量。少至六个额外的重复序列就可导致γ原点完全失去功能。然而，这种含有六个额外重复序列的无活性γ原点质粒，当置于野生型π蛋白产生不足的菌株中或置于几种拷贝增加突变型π蛋白存在的情况下时，是有功能的。基于这些观察结果，我们提出，由π蛋白与γ原点处的七个22-bp直接重复序列结合形成的核蛋白结构在体内能够相互偶联，并且在这种偶联的原点处复制起始被阻止。为支持这种复制控制模型，我们通过电子显微镜分析证明，π蛋白有能力使两个含有γ原点序列的DNA分子缔合，并且还表明π增强了DNA连接酶催化的含有γ原点的DNA片段的多聚化。
