NICKING ACTIVITY OF TRWC DIRECTED AGAINST THE ORIGIN OF TRANSFER OF THE INCW PLASMID R388
NICKING ACTIVITY OF TRWC DIRECTED AGAINST THE ORIGIN OF TRANSFER OF THE INCW PLASMID R388
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DOI：
10.1006/jmbi.1994.0065
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发表时间：
1995-02-10
影响因子：
5.6
通讯作者：
DELACRUZ, F
中科院分区：
文献类型：
--
作者：
LLOSA, M;GRANDOSO, G;DELACRUZ, F
广泛宿主范围质粒R388的接合DNA转移需要TrwC。纯化的蛋白质显示体外DNA解旋酶活性。在此，我们报告它也具有体外oriT-内切核酸酶活性：TrwC在Mg 2+存在下特异性地切割含有oriT的超螺旋质粒DNA，并且在用SDS处理后可以观察到切割的DNA。切口DNA的测序显示R388 oriT序列上较低DNA链的特异性中断。对切口的5'和3'端进行定位。5'端不能被T4多核苷酸激酶磷酸化，表明与TrwC共价结合。oriT中缺失的集合的分析表明，紧邻nic位点周围的核苷酸序列对于切口反应是重要的，但不是唯一的基本特征。R388 nic位点与之前发表的nic DNA序列的比较表明，IncF、IncN和IncW质粒形成了一个相关nic位点家族。在这项工作的过程中，我们还证明了Sequenase（TM）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位点的一些差异。
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.