MULTIPLE DNA CONFORMATIONAL-CHANGES INDUCED BY AN INITIATOR PROTEIN PRECEDE THE NICKING REACTION IN A ROLLING CIRCLE REPLICATION ORIGIN
MULTIPLE DNA CONFORMATIONAL-CHANGES INDUCED BY AN INITIATOR PROTEIN PRECEDE THE NICKING REACTION IN A ROLLING CIRCLE REPLICATION ORIGIN
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DOI：
10.1006/jmbi.1994.1242
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发表时间：
1994-04-08
影响因子：
5.6
通讯作者：
HORIUCHI, K
中科院分区：
文献类型：
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作者：
HIGASHITANI, A;GREENSTEIN, D;HORIUCHI, K
丝状噬菌体f1的正链DNA复制的核心起点与起始蛋白（gpII）结合，随后在正链中引入特异性切口。核心起源由切口区域和结合区域组成。gpII的结合分两步进行，形成结合中间体（复合物I）和用于切口的功能复合物（复合物II）。使用循环排列的DNA片段的凝胶阻滞实验和通过电子显微镜直接可视化的结果表明，GPII诱导连续弯曲的结合区域内形成的复合物。我们发现，gpII结合诱导双链体熔化的切口区域使用高锰酸钾修饰的未配对的胸苷残基作为探针熔化。起源结合发生在DNA和Mg 2+的超螺旋的情况下，而双链体熔化需要超螺旋DNA，但不是Mg 2+。缺失分析表明，假设形成一个十字形周围的切口网站是没有必要的熔化或切口。gpII的突变导致双链体熔化和切口的刺激，而没有显示出明显的影响弯曲。这表明熔化的机制涉及gpII和切口区域之间的局部相互作用。此外，使用合成的寡核苷酸底物，我们表明，切口反应有效地发生时，切口区域是单链和结合区域是双链的。这些结果表明，切口反应之前，由一系列有序的蛋白质诱导的DNA构象变化：连续弯曲的起源后，gpII结合，然后由双链熔融，需要负超螺旋。
The core origin for plus strand DNA replication of filamentous bacteriophage f1 binds the initiator protein (gpII), which subsequently introduces a specific nick in the plus strand. The core origin consists of a nicking region and a binding region. The binding of gpII occurs in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). Results of gel retardation experiments using circularly permuted DNA fragments and direct visualization by electron microscopy show that gpII induces successive bends within the binding region upon formation of the complexes. We show that gpII binding induces duplex melting in the nicking region using KMnO4modification of unpaired thymidine residues as a probe for melting. Origin binding occurred in the absence of superhelicity of DNA and Mg2+, whereas duplex melting required superhelical DNA, but not Mg2+. Deletion analyses indicated that hypothetical formation of a cruciform around the nicking site is not necessary for either melting or nicking. A mutation in gpII resulted in stimulation of duplex melting and nicking without showing obvious effects on bending. This suggests that the mechanism of melting involves local interaction between gpII and the nicking region. Furthermore, using synthetic oligonucleotide substrates, we show that the nicking reaction takes place efficiently when the nicking region is single-stranded and the binding region is double-stranded. These results indicate that the nicking reaction is preceded by an ordered series of protein induced DNA-conformational changes: successive bending of the origin upon gpII binding, followed by duplex melting that requires negative superhelicity.