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首页> 外文期刊>Molecular and Cellular Biology >Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.
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Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

机译：全位点重组过程中Flp重组酶的活性位点组装和DNA切割模式。

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A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.
机译：半位点底物和Flp（整合酶家族的一种位点特异性重组酶）的台阶突变突变体的组合早先揭示了半位点重组反应的以下特征。 （i）通过共享来自蛋白质的至少两个单体的催化残基来组装Flp活性位点。 （ii）Flp单体不裂解与其结合的半位（DNA顺式裂解）;相反，它切割由第二个Flp单体结合的半位（DNA反式切割）。对于Lambda整合酶（Int蛋白）（Int家族的原型成员），在与自杀性attL底物的反应中已观察到两个活性位点突变体之间的催化互补。类似于Flp，该观察结果强烈暗示了共享的活性位点和反式DNA切割。然而，与线性自杀attB底物和合成的霍利迪结的反应与顺式比与反式DNA切割更相容。这些Int结果要么与Int家族中常见的主动位点组装模式相矛盾，要么挑战Flp半位点作为正常全位点底物模拟物的有效性。我们设计了一种策略来分析Flp单体在完整位点之间的催化互补。我们发现全位反应遵循共享的活性位范式和DNA裂解的反式模式。这些结果表明，在Int家族中，重组的整体化学机理是通过重组酶单体之间的一种以上物理相互作用方式实现的。

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