Dixon J E, Sadowski P D
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
J Mol Biol. 1993 Dec 5;234(3):522-33. doi: 10.1006/jmbi.1993.1608.
The FLP site-specific recombinase is encoded by the two micron circle, an endogenous plasmid of Saccharomyces cerevisiae. FLP-mediated recombination in vitro proceeds via a short-lived Holliday (chi) intermediate. We have made a synthetic chi structure containing two FLP recognition target (FRT) sequences in order to investigate resolution by purified FLP protein. We found that incubation of this model substrate with FLP generated two pairs of linear products in equal quantities. Thus, resolution was equally likely to occur in either direction. Alteration of FLP binding sites, so as to inhibit binding, affected the direction of resolution; cleavage was reduced adjacent to the altered binding site. The overall efficiency of resolution increased when one FLP binding site was mutated. In investigating the series of mutated chi structures we found that resolution requires only two intact FLP binding sites. However, the non-specific protein-DNA interaction of additional FLP molecules may also be required. Thus, resolution is more tolerant of the loss of FLP binding sites than is the complete recombination reaction.
FLP位点特异性重组酶由酿酒酵母的内源性质粒双微体环编码。体外FLP介导的重组通过短暂存在的霍利迪（χ）中间体进行。为了研究纯化的FLP蛋白的拆分作用，我们构建了一个含有两个FLP识别靶点（FRT）序列的合成χ结构。我们发现，将该模型底物与FLP一起温育会产生等量的两对线性产物。因此，拆分在两个方向上发生的可能性相同。改变FLP结合位点以抑制结合，会影响拆分方向；在改变的结合位点附近切割减少。当一个FLP结合位点发生突变时，拆分的总体效率提高。在研究一系列突变的χ结构时，我们发现拆分仅需要两个完整的FLP结合位点。然而，可能还需要额外的FLP分子的非特异性蛋白质-DNA相互作用。因此，与完全重组反应相比，拆分对FLP结合位点缺失的耐受性更强。
