The traY Gene Product and Integration Host Factor Stimulate Escherichia coli DNA Helicase I-catalyzed Nicking at the F Plasmid oriT(*)
The traY Gene Product and Integration Host Factor Stimulate Escherichia coli DNA Helicase I-catalyzed Nicking at the F Plasmid oriT(*)
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traY 基因产物和整合宿主因子刺激大肠杆菌 DNA 解旋酶 I 催化 F 质粒 oriT 上的切口(*)
DOI：
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发表时间：
1995
影响因子：
4.8
通讯作者：
S. W. Matson
中科院分区：
文献类型：
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作者：
W. Nelson;M. Howard;Jonathan A. Sherman;S. W. Matson
质粒共轭转移是通过在质粒转移起始点（oriT）中引入位点和链特异性缺口而开始的。遗传学研究表明，缺口的形成依赖于traI和traY基因。然而，在体外缺乏traY基因产物（TraYp）的情况下，高度纯化的traI基因产物TraIp以位点和链特异性的方式进行转录(Matson， S. W., and Morton， B. S. (1991) J. Biol。化学，266,16232-16237)。oriT区的分析揭示了TraYp和宿主蛋白整合宿主因子（IHF）的结合位点。为了探索oriT中可能发生的相互作用，将高纯度的TraIp、TraYp和IHF与含有oriT的DNA底物一起培养。在一个同时需要TraYp和IHF的反应中，TraIp对缺口反应的催化作用明显增强。此外，当反应中存在IHF和TraYp时，TraIp能够刻出含有线性orit的双链DNA底物；这样的基板不是由TraIp单独切割的。超螺旋切口反应和线性切口反应的单个蛋白质浓度要求相似，反应发生的速度相同，表明它们在生物化学上是相同的。在切口分析中，TraYp和IHF的浓度产生一半最大活性，与报道的oriT中IHF和TraYp结合位点的K值相比较。这些数据，再加上随附报告中提供的数据，表明TraYp和IHF相互独立结合，与oriT形成核蛋白复合物，可以被TraIp识别和标记。
F plasmid conjugative transfer is initiated by the introduction of a site- and strand-specific nick within the plasmid origin of transfer (oriT). Genetic studies have shown nick formation to be dependent on both the traI and traY genes. However, highly purified TraIp, the traI gene product, nicks oriT in a site- and strand-specific manner in the absence of the traY gene product (TraYp) in vitro (Matson, S. W., and Morton, B. S. (1991) J. Biol. Chem. 266, 16232-16237). Analysis of the oriT region has revealed binding sites for TraYp and the host protein integration host factor (IHF). To explore possible interactions occurring at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate. A marked enhancement of the nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF. In addition, TraIp was able to nick a linear oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone. Individual protein concentration requirements for the supercoiled and linear nicking reactions were similar, and the reactions occurred at equal velocity, suggesting that they are biochemically identical. Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported K values for the IHF and TraYp binding sites in oriT. These data, coupled with data presented in the accompanying report, suggest that TraYp and IHF bind independent of one another, forming a nucleo-protein complex with oriT that can be recognized and nicked by TraIp.