Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.
Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.
复制标题
体外 DNA 复制的终止：枯草芽孢杆菌复制终止子蛋白的两个二聚体之间以及与终止子位点之间立体特异性相互作用的要求，以引发极性反解旋酶和叉阻抗。
DOI：
10.1002/j.1460-2075.1995.tb07038.x
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Bastia,D
中科院分区：
文献类型：
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作者：
Sahoo,T;Mohanty,BK;Patel,I;Bastia,D
在枯草芽孢杆菌中，一种亚单位摩尔的二聚体复制终止蛋白（RTP）催化了序列特异性复制末端DNA复制的终止。RTP已经成为研究终止分子机制的一个有吸引力的蛋白质，因为它的晶体结构现在已经解决了，以前缺乏体外复制系统的问题已经很大程度上被我们的发现所克服，在体内和体外，该蛋白质在已经研究得很好的革兰氏阴性大肠杆菌系统中终止复制。我们利用体外替代系统表明，只有当两个RTP二聚体合作结合到重叠的核心和包括末端的辅助序列时，RTP才能作为大肠杆菌dna解旋酶的极性对解旋酶。核心序列本身结合RTP的一个二聚体，但不引起对旋酶活性。两个RTP二聚体与串联头尾核心重复序列的结合也不会引起对旋酶活性，因此表明两个RTP二聚体之间以及与终止位点的特定立体化学相互作用对于终止至关重要。RTP阻断含有异双工区的DNA底物的解绕，这些异双工区包括末端，长度范围约为50至1000 bp。因此，该蛋白阻断了真正的解旋酶催化的解绕，而不仅仅是解旋酶在DNA上的易位。
The termination of DNA replication at a sequence‐specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well‐studied Gram‐negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co‐operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head‐to‐tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase‐catalyzed unwinding rather than just the translocation of the helicase on DNA.