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首页> 外文期刊>Journal of Molecular Biology >The Efficiency of Mispaired Ligations by lambda Integrase is Extremely Sensitive to Context.
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The Efficiency of Mispaired Ligations by lambda Integrase is Extremely Sensitive to Context.

机译：lambda Integrase导致错配连接的效率对上下文极为敏感。

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The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.
机译：噬菌体λ的整合酶（Int）是酪氨酸重组酶家族的一个经过充分研究的代表，其定义特征是DNA裂解/连接反应的两个连续对，这些反应通过3'磷酸酪氨酸共价中间物进行，先形成第一个形式，然后解析一个霍利迪交界处重组中间体。我们设计了一种利用在一个Int目标位点形成DNA发夹结构的方法来捕获在另一个目标位点处的Int切割的方法，从而揭示了原本无法检测到的切割和连接的重复循环。使用该测定法和其他方法比较野生型Int和突变体（R169D）在形成适当的二聚体/四聚体界面方面存在缺陷，我们发现野生型Int而非“ R169D”对“底部链” DNA的切割效率是对“顶部链”切割位点的碱基对非常敏感，距离7个碱基对。我们表明，这与以下发现有关：涉及到错配碱基的连接的发夹形成对于R169D而言要比野生型Int快得多，但仅在多聚体复合物的情况下才如此。在解析霍利迪（Holliday）连接重组中间体期间，野生型Int（而非R169D）对连接位点的同源性非常敏感。从这些结果中可以找到一个长期的见解，那就是在霍利迪（Holliday）连接解析期间，四聚体Int复合物保持完整，直到完成产物螺旋的连接为止。这与其中第二对DNA切割是重组复合物溶解的触发因素的模型形成对比。

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