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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Role of the double-strand origin cruciform in pT181 replication.
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Role of the double-strand origin cruciform in pT181 replication.

机译：双链起源十字形在pT181复制中的作用。

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摘要

pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBO J. 16, 4456-4566). In this study, we have tested the requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested for replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR II disrupted were less efficiently relaxed than the wild type by RepC. However, in conjunction with IR II disruption, modification of the tight RepC binding site IR III blocked replication. These results define two key elements of the pT181 initiation mechanism--the IR II conformation and the RepC binding site (IR III)--and they indicate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO.
机译：pT181是一种来自金黄色葡萄球菌的小滚环质粒，其启动子蛋白RepC融化了质粒的双链起源（DSO），并挤出了一个包含IR II的十字形，这是一个位于起始切口位点旁的回文。我们假设启动需要十字形，为复制体的组装提供了单链区域（R. Jin等，1997，EMBO J. 16，4456-4566）。在这项研究中，我们通过破坏IR II回文的对称性或增加其长度来测试十字形挤压的要求。使用RepC反式测试了修饰的DSO的复制。令人惊讶的是，IR II对称性的破坏对修饰的DSO的复制或竞争性没有可检测的影响，尽管具有RepC的IR II被破坏的质粒比野生型的松弛效率低。但是，结合IR II破坏，紧密RepC结合位点IR III的修饰会阻止复制。这些结果定义了pT181起始机制的两个关键要素-IR II构象和RepC结合位点（IR III）-并且它们表明pT181复制起始具有足够的鲁棒性，能够补偿PT181构型的重大修饰。 DSO。

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