Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38
Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38
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DOI：
10.1128/jb.01695-06
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发表时间：
2007-03-01
影响因子：
3.2
通讯作者：
Itoh, Tateo
中科院分区：
文献类型：
--
作者：
Aoki, Kazuteru;Shinohara, Miki;Itoh, Tateo
质粒 ColE2-P9 Rep 蛋白特异性结合同源复制起点以启动 DNA 复制。质粒 ColE2-P9 和 ColE3-CA38 的复制子密切相关，尽管 Rep 蛋白对起点的作用是质粒特有的。先前的嵌合体分析鉴定出Rep蛋白中的两个区域A区和B区以及起源中的两个位点α和P作为特异性决定簇，并表明当A区位点α对和区域B位点β对的每个组成部分均源自同一质粒时，质粒DNA复制是有效的。也表明复制特异性主要由A区和α位点决定。通过使用电泳迁移率变动分析，我们证明了 B 区和位点 β 对于稳定的 Rep 蛋白起点结合起着关键作用，此外，ColE2 Rep 蛋白该区域中的 284-Thr 和 ColE3 Rep 蛋白相应的 293-Trp 主要决定 Rep 起点结合特异性。另一方面，A区和α位点参与了ut位点周围几个核苷酸残基的有效解旋，尽管它们不参与Rep蛋白与起点的稳定结合。最后，我们讨论了 Rep 蛋白对涉及这些特异性决定簇的起源的作用如何导致质粒特异性复制起始。
The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and P, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site ut, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.