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首页> 外文期刊>Journal of Molecular Biology >NICKING ACTIVITY OF TRWC DIRECTED AGAINST THE ORIGIN OF TRANSFER OF THE INCW PLASMID R388
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NICKING ACTIVITY OF TRWC DIRECTED AGAINST THE ORIGIN OF TRANSFER OF THE INCW PLASMID R388

机译：根据INCW质粒R388的转移原点，TRWC的结网活性

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摘要

TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity Here we report that it also has in vitro oriT-endonuclease activity: TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase(TM) Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems. [References: 49]
机译：TrwC是广泛宿主范围质粒R388的结合DNA转移所必需的。纯化的蛋白质显示出体外DNA解旋酶活性在这里我们报道它也具有体外oriT核酸内切酶活性：TrwC在Mg2 +存在的情况下特异地刻痕含oriT的超螺旋质粒DNA，用SDS处理后可以看到有刻痕的DNA。有缺口的DNA的测序显示R388 oriT序列上的下部DNA链有特定的中断。刻痕的5'和3'末端均已映射。 T4多核苷酸激酶的5'末端不易被磷酸化，表明与TrwC共价结合。对oriT缺失集合的分析表明，直接位于nic位点周围的核苷酸序列对于切口反应很重要，但不是唯一的必需特征。 R388 nic位点与以前发布的nic DNA序列的比较表明，IncF，IncN和IncW质粒形成了一系列相关的nic位点。在这项工作的过程中，我们还证明了Sequenase™2.0版DNA聚合酶的末端转移酶活性（尚未记录），这可以解释以前在其他系统中映射的nic站点中的某些差异。 [参考：49]

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