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首页> 外文期刊>Journal of Molecular Biology >CHARACTERIZATION OF THE INTERACTION BETWEEN THE LAMBDA INTASOME AND ATTB
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CHARACTERIZATION OF THE INTERACTION BETWEEN THE LAMBDA INTASOME AND ATTB

机译：Lambda病原体与ATTB之间相互作用的表征

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摘要

Bacteriophage lambda DNA integrates into the chromosome of Escherichia coli by first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor. This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration. This study characterizes the weak interaction of the intasome and attB. The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome, is given additional support by showing that the concentration of phage attachment site and not attB determines the optimal concentration of proteins for integration. The value of the dissociation constant of the complex formed between the intasome and attB is determined in two different ways. First, the rate of the integration reaction is measured as a function of the attB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recombination reaction is inhibited by the introduction of varying amounts of a second attB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and altered attB in this experiment. The two methods agree and give a dissociation constant of approximately 300 nM. attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture. TM value of the dissociation constants are considerably lower when a mutant integrase, IntE174K, is used. This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor. The inhibition constants also show dependence on the exact sequence of the inhibiting attB. Possible implications of this dependence are discussed. (C) 1995 Academic Press Limited [References: 43]
机译：噬菌体λDNA通过首先在噬菌体DNA上的噬菌体附着位点形成整合酶Int和整合宿主因子，从而整合到大肠杆菌的染色体中。该整合体在宿主染色体上搜索细菌附着位点（attB），然后编排两个顺序的链交换反应以实现整合。这项研究的特点是intasome和attB的弱相互作用。整合所需的所有蛋白质都被内含体带入反应位点的假说通过显示噬菌体附着位点而不是attB的浓度决定了整合所需的最佳蛋白质浓度而得到了进一步的支持。以两种不同的方式确定在染色体和attB之间形成的复合物的解离常数的值。首先，根据attB DNA浓度测量整合反应的速率。饱和常数反映了配合物的解离常数。其次，通过引入不同量的第二attB来抑制重组反应，第二attB的序列变化会阻止与该位点的重组。在该实验中，抑制常数反映了Intasome的解离常数和改变的attB。两种方法一致，并给出约300 nM的解离常数。 attB包含两个intasome的核心结合位点;结果表明，两者都是有效捕获所必需的。使用突变型整合酶IntE174K时，解离常数的TM值要低得多。对核心位点的亲和力增加可以解释IntE174K在没有整合宿主因子的情况下如何发挥作用。抑制常数也显示出对抑制attB的确切序列的依赖性。讨论了这种依赖性的可能含义。 （C）1995 Academic Press Limited [参考：43]

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