Lee E H, Kornberg A, Hidaka M, Kobayashi T, Horiuchi T
Department of Biochemistry, Stanford University, CA 94305-5307.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9104-8. doi: 10.1073/pnas.86.23.9104.
Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E. coli chromosome (oriC) in a purified enzyme system. Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it. The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP. Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction. From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side. Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions.
大肠杆菌中复制终止共有序列（ter）的鉴定以及ter结合蛋白（TBP）的分离，使我们能够在纯化的酶系统中测试它们对从大肠杆菌染色体唯一复制起点（oriC）起始的复制叉的影响。当提供纯化的TBP结合ter时，复制在特定方向上会受到严重阻碍。复制复合物内的阻断靶点现在可以归因于当dnaB解旋酶遇到与TBP结合的ter时，无法分离双链。其他在DNA上移位并沿与dnaB蛋白相反方向置换链的解旋酶，如rep和uvrD蛋白，也会被阻断，但只有当与TBP结合的ter在另一个方向上时才会被阻断。从这些结果中，我们推断ter的方向赋予了位于其上的TBP特定的极性，使得解旋酶从一侧面对TBP时会被阻断，但当面对其另一侧时，大概是通过置换TBP，解旋酶可以发挥作用。因此，定向的TBP-ter复合物的内在性质是阻碍解旋酶的原因，而不是任何蛋白质-蛋白质相互作用。
