Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins.
Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins.
复制标题
质粒 R6K 复制的负控制：复制起点分子间偶联的可能作用。
DOI：
10.1073/pnas.86.20.7942
复制
发表时间：
1989
影响因子：
11.1
通讯作者：
Helinski,DR
中科院分区：
文献类型：
--
作者：
McEachern,MJ;Bott,MA;Tooker,PA;Helinski,DR
复制启动器pi蛋白的γ起始结合位点由7个22碱基对（bp）的直接重复序列组成，以前被证明是质粒R6K复制所必需的，如果以反式形式提供，也可以作为大肠杆菌细胞中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pi蛋白的能力，但不能通过增加细胞内pi水平来克服。在伽马起始点附近插入第二个重复簇也能显著抑制复制。这种影响的严重程度取决于位置，方向和重复出现在额外的簇的数量。只要多重复6次，就会导致完全无功能的γ起始。然而，这种含有6个额外重复序列的无活性γ源质粒在野生型pi蛋白生产不足的菌株中或在几种复制突变的pi蛋白中存在时仍具有功能。在这些观察的基础上，我们提出pi蛋白在γ起点与7个22 bp直接重复序列结合形成的核蛋白结构能够在体内相互耦合，并且在这种耦合起点上阻止复制起始。为了支持这种复制控制模型，我们通过电子显微镜分析证明了pi蛋白具有结合两个含有γ起始序列的DNA分子的能力，并且还表明pi增强了DNA连接酶催化的含有γ起始序列的DNA片段的多聚化。
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.