Chen J W, Yang S H, Jayaram M
Department of Microbiology, University of Texas, Austin 78712.
J Biol Chem. 1993 Jul 5;268(19):14417-25.
The Arg191-His305-Arg308 (the RHR triad) and Tyr343 of Flp site-specific recombinase correspond to the invariant tetrad residues of the integrase family of proteins. Flp mutants altered at these positions are blocked at the strand cleavage or the strand exchange step of recombination. Hybrid half-site-recombinase complexes formed by step-arrest mutants of Flp have revealed that an Flp monomer occupying a half-site does not cleave that half-site but rather cleaves a half-site occupied by a second Flp monomer. This trans-DNA cleavage is neatly accommodated by a model in which an Flp active site is assembled by contribution of amino acid residues from at least two protein monomers. Using a combination of wild type Flp, single, double, and triple step-arrest Flp mutants, critical predictions of the fractional active-site model have been verified. First, a wild type monomer paired with an RHR triad-Tyr343 double mutant is a catalytically inactive combination. Second, each pairwise combination of a single, double, or triple RHR mutant with Flp (Y343F) yields approximately equivalent levels of catalytic complementation. Half-site to half-site and half-site to full-site crosses suggest that execution of a strand transfer event within a half-site and between a half-site and a full site requires dimeric and tetrameric Flp configurations, respectively.
Flp位点特异性重组酶的Arg191-His305-Arg308（RHR三联体）和Tyr343对应于整合酶家族蛋白质的不变四联体残基。在这些位置发生改变的Flp突变体在重组的链切割或链交换步骤中受阻。由Flp的逐步阻滞突变体形成的杂交半位点-重组酶复合物表明，占据一个半位点的Flp单体不会切割该半位点，而是切割由第二个Flp单体占据的半位点。这种反式DNA切割可以通过一个模型很好地解释，在该模型中，Flp活性位点是由至少两个蛋白质单体的氨基酸残基共同作用组装而成的。通过结合野生型Flp、单步、双步和三步阻滞Flp突变体，分数活性位点模型的关键预测得到了验证。首先，与RHR三联体-Tyr343双突变体配对的野生型单体是一种无催化活性的组合。其次，单个、双个或三个RHR突变体与Flp（Y343F）的每对组合产生大致相当水平的催化互补。半位点到半位点以及半位点到全位点的交叉表明，在一个半位点内以及在一个半位点和一个全位点之间进行链转移事件分别需要二聚体和四聚体Flp构型。
