Segall A M, Nash H A
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892-4034, USA.
Genes Cells. 1996 May;1(5):453-63. doi: 10.1046/j.1365-2443.1996.d01-254.x.
In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo, recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5-10-fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro.
We find that IHF can stimulate the in vitro recombination between two attLs that are modified to be defective in one of the high affinity binding sites for Int, P'1. With such substrates, the efficiency of IHF-stimulated recombination is comparable to that seen in vivo. The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo, but that some unknown factor or condition permits it to operate on wild-type as well as mutated attL sites.
The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P'1 site. In addition to demonstrating the architectural flexibility of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.
在噬菌体λ的生命周期中，整合酶（Int）蛋白在两组不同的DNA底物之间进行重组：整合时的attP和attB，切除时的attL和attR。在每种情况下，这两个伙伴在结构上彼此差异很大，它们之间的重组反应实际上是不可逆的。为了进行比较，我们研究了Int介导的两个相同attL位点之间的重组。在体外和体内，仅Int就能低效地介导两个attL位点之间的重组。但是，虽然整合宿主因子（IHF）能在体内将重组刺激5至10倍（达到切除和整合的水平），但在体外标准条件下未观察到这种刺激作用。
我们发现，对于在Int的一个高亲和力结合位点P'1上被修饰成有缺陷的两个attL之间的体外重组，IHF能够起到刺激作用。对于这样的底物，IHF刺激的重组效率与在体内观察到的相当。该反应的要求使其与其他λ重组途径区分开来，几种突变Int蛋白的表现也是如此。细胞内质粒上attL位点的重组表明，该途径在体内是有效的，但一些未知因素或条件允许它在野生型以及突变的attL位点上发挥作用。
本研究中描述的重组途径显然使用了一种独特的attL结构，这种结构需要IHF弯曲，并且会被结合在P'1位点的Int抑制。除了证明λ系统的结构灵活性外，该途径对于将链交换化学的基本要求与赋予方向性的特征区分开来应该是一种有价值的资源。
