Hiasa H, Marians K J
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1994 Oct 28;269(43):26959-68.
Minichromosome plasmid DNA templates containing oriC and two Ter sites, oriented as they are on the Escherichia coli chromosome, have been used to study the role of Tus in termination of bidirectional replication. In replication reactions reconstituted with purified proteins where it could be demonstrated that each active template was replicating bidirectionally, Tus was required to prevent extensive overreplication. In the presence of Tus, both replication forks terminated DNA synthesis at one or the other Ter site in an apparent stepwise manner. First, the progress of one replication fork was arrested by a properly oriented Tus-Ter complex. Then, either because of steric hindrance resulting from the stalled replication machinery of the first fork or because of the formation of a branched DNA structure, the progression of the second opposing fork was halted at the same site on the DNA template. In the absence of Tus, overreplication required DNA ligase and arose via a template strand-switching mechanism. Thus, the role of Tus in E. coli is more likely to prevent overreplication rather than to ensure accurate termination.
含有oriC和两个Ter位点的微型染色体质粒DNA模板，其方向与它们在大肠杆菌染色体上的方向相同，已被用于研究Tus在双向复制终止中的作用。在用纯化蛋白重建的复制反应中，可以证明每个活性模板都在双向复制，Tus是防止广泛过度复制所必需的。在Tus存在的情况下，两个复制叉都以明显的逐步方式在一个或另一个Ter位点终止DNA合成。首先，一个正确定向的Tus-Ter复合物阻止了一个复制叉的前进。然后，要么是由于第一个复制叉停滞的复制机制造成的空间位阻，要么是由于形成了分支DNA结构，第二个相对的复制叉在DNA模板的同一位点停止前进。在没有Tus的情况下，过度复制需要DNA连接酶，并通过模板链交换机制产生。因此，Tus在大肠杆菌中的作用更可能是防止过度复制，而不是确保准确终止。
