Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.
Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.
复制标题
酶催化 DNA 解旋：大肠杆菌代表蛋白的研究。
DOI：
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发表时间：
1979
影响因子：
11.1
通讯作者：
M. Gefter
中科院分区：
文献类型：
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作者：
G. Yarranton;M. Gefter
噬菌体varphiX174的复制型（RF）I DNA的体外复制需要噬菌体诱导的顺反子A（cisA）蛋白、宿主rep蛋白、DNA结合蛋白、ATP和DNA聚合酶III加上复制因子。rep蛋白是一种单链DNA依赖性ATP酶。在本文中，我们表明，varphiX174 RF I DNA切割的cisA蛋白作为一个双链体DNA辅因子的REP蛋白ATP酶活性，提供的DNA结合蛋白的存在。在后一种反应中，双链体DNA被rep蛋白解绕，伴随着ATP的水解。ATP水解、DNA解旋和DNA合成的程度与存在的DNA结合蛋白的量成比例。每对解旋的碱基对水解两个ATP分子。我们建议，强制性要求的cisA蛋白的解旋varphiX174 RF I DNA不是简单地由于其核酸内切酶活性，而是由于它提供了一个网站的约束力的rep蛋白。在DNA结合蛋白存在下，但在cisA蛋白不存在下，当一条链延伸以在双链体之前产生单链前导区时，rep蛋白解旋双链体DNA。我们发现，rep蛋白只在5 '到3'的方向上沿着前导单链沿着易位，然后侵入双链体DNA。rep蛋白对易位和解旋具有方向特异性。提出了一个模型来解释由rep蛋白催化的DNA解旋的机制。
Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.