Bor Y C, Bushman F D, Orgel L E
Salk Institute for Biological Studies, San Diego, CA 92186-8500, USA.
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10334-8. doi: 10.1073/pnas.92.22.10334.
Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.
1型人类免疫缺陷病毒（HIV-1）的互补DNA（cDNA）与靶DNA的整合会受到靶标构象的强烈影响。例如，在体外，整合有时在含有序列导向弯曲或由结合蛋白引起的DNA扭曲的靶DNA中更受青睐。我们通过研究整合到两种特性明确的蛋白质-DNA复合物中，分析了DNA弯曲的影响：与噬菌体整合宿主因子（IHF）位点结合的大肠杆菌整合宿主因子（IHF）蛋白，以及与淋巴增强因子（LEF）位点结合的人类淋巴增强因子（LEF）的DNA结合结构域。此前已有报道称，这两种蛋白质都会使DNA弯曲约140度。IHF的结合大大提高了体外在IHF位点热点处的整合效率。我们分析了一系列突变体，其中IHF位点在最突出的热点处进行了修饰。我们发现，每个变体在IHF结合后仍表现出增强的整合。显然，局部序列对于形成IHF热点并不关键。LEF的结合并未产生整合的优先位点。根据两种蛋白质-DNA复合物的不同构象，可以解释IHF和LEF结合的不同效果。
