Han Y W, Gumport R I, Gardner J F
Department of Microbiology, College of Medicine, University of Illinois at Urbana-Champaign 61801.
EMBO J. 1993 Dec;12(12):4577-84. doi: 10.1002/j.1460-2075.1993.tb06146.x.
Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.
噬菌体λ的位点特异性重组始于形成称为“整合体”的高阶蛋白质-DNA复合物，随后是一系列步骤，包括初始DNA切割、顶链交换、分支迁移和底链交换，以产生重组产物。在切除性重组过程中形成的一种整合体（attL复合物）由噬菌体编码的整合酶（Int）、整合宿主因子（IHF）和一种重组底物attL DNA组成。Int是催化性重组酶，具有两个不同的DNA结合结构域。当存在IHF时，Int结合attL DNA中的两种类型的位点，即三个臂型位点（P'123）和发生相互链交换的核心型位点（B和C'）。Int的Tyr342残基在链切割过程中作为亲核试剂，并通过磷酸酪氨酸键与DNA共价连接。已经使用attL自杀底物和在重组酶整合酶家族中保守残基处含有氨基酸取代的突变蛋白进行了链切割的体外互补试验。我们证明，至少需要两个Int单体才能形成在B位点进行切割的具有催化活性的物种。活性位点可能由两个Int单体形成。
