Llosa M, Grandoso G, de la Cruz F
Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
J Mol Biol. 1995 Feb 10;246(1):54-62. doi: 10.1006/jmbi.1994.0065.
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 Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.
TrwC是广宿主范围质粒R388接合DNA转移所必需的。纯化后的蛋白表现出体外DNA解旋酶活性。在此我们报道它还具有体外oriT内切核酸酶活性。TrwC在Mg2+存在的情况下特异性切割含oriT的超螺旋质粒DNA，经SDS处理后可观察到切口DNA。对切口DNA进行测序显示R388 oriT序列上的下链有特异性中断。确定了切口的5'端和3'端位置。5'端不能被T4多核苷酸激酶磷酸化，表明它与TrwC存在共价结合。对oriT中一系列缺失进行分析表明，紧邻切口位点的核苷酸序列对切口反应很重要，但不是唯一的必需特征。将R388切口位点与先前发表的切口DNA序列进行比较表明，IncF、IncN和IncW质粒形成了一个相关切口位点家族。在这项工作过程中，我们还证明了Sequenase Version 2.0 DNA聚合酶存在一种尚未记录的末端转移酶活性，这可能解释了其他系统中先前定位的切口位点存在的一些差异。
