Role of the double-strand origin cruciform in pT181 replication
Role of the double-strand origin cruciform in pT181 replication
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DOI：
10.1006/plas.2001.1535
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发表时间：
2001-09-01
期刊：
影响因子：
2.6
通讯作者：
Novick, RP
中科院分区：
文献类型：
--
作者：
Jin, RZ;Novick, RP
pT181 是来自金黄色葡萄球菌的小滚环质粒，其起始蛋白 RepC 熔化质粒的双链起点 (DSO)，并挤出一个涉及 IR II 的十字形，即起始切口位点侧翼的回文结构。我们假设十字形是启动所必需的，为复制体的组装提供单链区域(R.Jin等人，1997，EMBO J.16.4456-4566)。在本研究中，我们通过破坏 IR II 回文结构的对称性或增加其长度来测试十字形挤出的要求。测试了修改后的 DSO 与 RepC 的反式复制。相当令人惊讶的是，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到的影响，尽管 IR H 被破坏的质粒被 RepC 松弛的效率低于野生型。然而，与 IR II 破坏相结合，RepC 紧密结合位点 IR III 的修饰阻碍了复制。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 复制起始足够稳健，能够补偿 DSO 配置的显着修改。 (C) 2001 年学术出版社。
pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts die 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 H 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. (C) 2001 Academic Press.