Cleavage-dependent Ligation by the FLP Recombinase CHARACTERIZATION OF A MUTANT FLP PROTEIN WITH AN ALTERATION IN A CATALYTIC AMINO ACID*
Cleavage-dependent Ligation by the FLP Recombinase CHARACTERIZATION OF A MUTANT FLP PROTEIN WITH AN ALTERATION IN A CATALYTIC AMINO ACID*
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发表时间：
1995
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影响因子：
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通讯作者：
Xudong Zhu;P. Sadowski
中科院分区：
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作者：
Xudong Zhu;P. Sadowski
酿酒酵母的2（cid：109）M质粒的FLP重组酶属于重组酶的整合酶家族，其成员共有四个绝对保守的残基（Arg-191、His-305、Arg-308和Tyr-343）。我们研究了突变蛋白FLP R308 K，其中308位的精氨酸残基被赖氨酸取代。尽管先前报道FLP R308 K在某些底物的连接中有缺陷（Pan，G.，Luetke，K.，和Sadowski，P. D.摩尔Cell. 13，3167-3175，1993 b），我们在这项工作中表明，该蛋白能够连接它可以切割的那些底物（切割依赖性连接活性）。FLP R308 K在体外重组和链交换中有缺陷。它能够在FLP识别靶位点（FRT位点）的两个切割位点之一进行链交换，但在另一个切割位点的链交换有缺陷。这些结果与野生型FLP仅在两个切割位点之一启动重组的模型一致。FLP R308 K可能在重组起始中有缺陷。
The FLP recombinase of the 2 (cid:109) M plasmid of Saccharomyces cerevisiae belongs to the integrase family of recombinases whose members have in common four absolutely conserved residues (Arg-191, His-305, Arg-308, and Tyr-343). We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine. Although FLP R308K was previously reported to be defective in ligation of certain substrates (Pan, G., Luetke, K., and Sadowski, P. D., Mol. Cell. Biol. 13, 3167–3175, 1993b), we show in this work that the protein is able to ligate those substrates that it can cleave (cleavage-dependent ligation activity). FLP R308K is defective in in vitro recombination and in strand exchange. It is able to carry out strand exchange at one of the two cleavage sites of the FLP recognition target site (FRT site), but is defective in strand exchange at the other cleavage site. These results are consistent with a model in which wild-type FLP initiates recombi- nation only at one of the two cleavage sites. FLP R308K may be defective in the initiation of recombination.