Gamma integrase complementation at the level of DNA binding and complex formation.
Gamma integrase complementation at the level of DNA binding and complex formation.
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DNA 结合和复合物形成水平上的伽玛整合酶互补。
DOI：
10.1128/jb.184.5.1385-1394.2002
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发表时间：
2002
影响因子：
3.2
通讯作者：
Landy,Arthur
中科院分区：
文献类型：
--
作者：
Nunes-Düby,SimoneE;Radman-Livaja,Marta;Kuimelis,RobertG;Pearline,RachelV;McLaughlin,LarryW;Landy,Arthur
DNAInt家族的位点特异性重组酶通过以头对头二聚体的形式结合在倒置的核心型λ位点上进行两次单链交换。每个原核糖体可以作为单体切割自己的位点(对于Cre重组酶)，或者它可以从其伴侣的反式体内招募酪氨酸形成一个复合的活性位点(对于FLP重组酶)。λInt催化结构域的晶体结构与这两种切割机制都是兼容的，但之前两项关于λ整合酶(Int)的生化研究产生的数据并不一致。支撑力和跨裂解分别来自对λ和HK022Ints的双特异性DNA底物检测和重组缺陷突变体之间的功能互补。这里提供的数据没有提供新的证据，但它们强烈表明，前面描述的互补结果不能用于支持反式切割机制。在这里，我们发现IntR212Q保留了一些残留的催化功能，但在与线形底物上的核心型DNA结合和形成高阶L端粒体结构方面受到了损害。精通结合的突变体IntY342F可以通过蛋白质-蛋白质相互作用稳定IntR212Q与核心型DNA的结合。同样，两个突变体的存在都促进了与臂和核心型DNA形成高阶Int复合体。这种互补先于裂解，因此排除了关于催化机制的任何结论。突变体λInts在双特异性核心自杀底物上对野生型HK022-Int同源位点(INCI)的跨核心刺激被证明不依赖于催化酪氨酸，但似乎与λInt突变体的核心结合亲和力成正比。
Site-specific recombinases of the λ Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer incis(as for Cre recombinase), or it may recruit the tyrosine from its partner intransto form a composite active site (as for Flp recombinase). The crystal structure of the λ Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on λ integrase (Int) generated data that were not in agreement. Support forcisandtranscleavage came from assays with bispecific DNA substrates for λ and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence forciscleavage, but they strongly suggest that the previously described complementation results cannot be used in support of atrans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-orderattL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (incis) by mutant λ Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the λ Int mutants.