Duggin I G, Andersen P A, Smith M T, Wilce J A, King G F, Wake R G
Department of Biochemistry, University of Sydney, NSW, 2006, Australia.
J Mol Biol. 1999 Mar 12;286(5):1325-35. doi: 10.1006/jmbi.1999.2553.
DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork.
在枯草芽孢杆菌染色体复制的终止阶段，DNA复制叉的停滞是由结合到分布于整个终止区的特定DNA终止序列（Ter位点）的复制终止蛋白（RTP）引起的。其他人提出的一个有吸引力的观点是，RTP-Ter复合物发挥功能的关键在于位于DNA上的RTP与接近复制叉的解旋酶之间的特异性相互作用。支持这一观点的是RTP的两个定点突变体的行为。它们似乎能正常结合Ter DNA，但通过体外大肠杆菌DnaB解旋酶和复制试验确定，它们在阻止复制叉方面无效。我们在此描述了一个在枯草芽孢杆菌中进行真正体内试验来评估RTP突变体阻止复制叉行为的系统。如预期的那样，之前研究的一个突变体RTP.Y33N在体内阻止复制叉方面无功能。但通过广泛分析，这个RTP突变体被证明在结合Ter DNA方面存在严重缺陷，这与预期相反。结合最近关于另一个突变体（RTP.E30K）的发现，可以得出结论，从RTP突变体的行为中还没有实质性证据支持用于阻止复制叉的RTP-解旋酶相互作用模型。在对RTP.Y33N当前工作的扩展中，我们测定了野生型（wt）RTP和另一个RTP突变体与各种终止序列形成的复合物的解离速率。功能性的wtRTP-TerI复合物相当稳定（半衰期为182分钟），这让人想起大肠杆菌Tus-Ter复合物的高度稳定性。更值得注意的是，由wtRTP和一个RTP双突变体（E39K.R42Q）与一些特定终止序列结合形成的复合物具有异常的稳定性。从对各种复合物体内阻止复制叉活性的测量结果可以得出结论，整个RTP-Ter复合物的稳定性（半衰期）不是阻止复制叉行为的首要决定因素；并且RTP-Ter复合物必须通过接近的复制叉的作用被主动破坏，或者RTP被移除。
