Dixon J E, Shaikh A C, Sadowski P D
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Mol Microbiol. 1995 Nov;18(3):449-58. doi: 10.1111/j.1365-2958.1995.mmi_18030449.x.
The Flp site-specific recombinase is encoded by the 2 micrometers plasmid Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges. Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site. It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (chi) structures to linear products. In this paper, we examined whether resolution of chi structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked chi structures to show that Flp resolves chi structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.
Flp位点特异性重组酶由酿酒酵母的2微米质粒编码，是重组酶整合酶家族的成员。与所有已研究的整合酶家族成员一样，Flp介导的重组分两步进行。首先，一对链交换产生一个类Holliday中间体；其次，该中间体通过另一对链交换被解析为重组产物。使用线性底物的实验证据表明，Flp的活性位点由两个Flp原体组成。一个与Flp识别靶位点（FRT位点）结合并激活可裂解的磷酸二酯键以进行切割。在突触复合物其他位置结合的另一个Flp分子（反式）提供执行切割的亲核酪氨酸，从而共价连接到切割位点的3'磷酸基团上。先前已表明，Flp能有效地将合成的、类Holliday（chi）结构解析为线性产物。在本文中，我们研究了Flp对chi结构的解析是否也通过反式切割机制发生。我们使用突变Flp蛋白的体外互补研究以及带切口的chi结构来表明Flp通过反式切割解析chi结构。我们提出了一个Flp介导重组的模型，该模型在链交换的初始步骤和解析步骤都纳入了反式切割。
