Kitts P A, Nash H A
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892.
Nucleic Acids Res. 1988 Jul 25;16(14B):6839-56. doi: 10.1093/nar/16.14.6839.
It has been proposed that phage lambda site-specific recombination proceeds via two independent strand exchanges: the first exchange forming a Holliday-structure which is then converted into complete recombinant products by the second strand exchange. If this hypothesis is correct, one should be able to trap the putative Holliday intermediate by preventing the second strand exchange. In this paper, we show that substitution of phosphorothioate for phosphate in one strand of a recombination site is an effective way to block recombination while permitting the accumulation of a novel structure. This effect is seen only when phosphorothioate is positioned at a point of potential cleavage by Int recombinase, demonstrating that the inhibition of strand exchange is highly specific. Analysis of the novel structure that accumulates in these reactions proves that it contains a Holliday joint. Holliday-structures can also be detected in unblocked recombinations but are present at very low levels. The characteristics of Holliday-structure formation that we describe substantiate the proposed recombination pathway.
有人提出，λ噬菌体位点特异性重组是通过两个独立的链交换进行的：第一次交换形成一个霍利迪结构，然后通过第二次链交换将其转化为完整的重组产物。如果这个假设是正确的，那么应该能够通过阻止第二次链交换来捕获假定的霍利迪中间体。在本文中，我们表明，在重组位点的一条链中用硫代磷酸酯取代磷酸酯是一种有效的方法，既能阻止重组，又能使一种新结构积累。只有当硫代磷酸酯位于Int重组酶潜在切割点时，才会出现这种效应，这表明链交换的抑制具有高度特异性。对这些反应中积累的新结构的分析证明它含有一个霍利迪连接体。在未受阻的重组中也能检测到霍利迪结构，但含量非常低。我们描述的霍利迪结构形成的特征证实了所提出的重组途径。
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