Grainge Ian, Pathania Shailja, Vologodskii Alexander, Harshey Rasika M, Jayaram Makkuni
Section of Molecular Genetics and Microbiology and the Institute of Cell and Molecular Biology, University of Texas at Austin, 78712, USA.
J Mol Biol. 2002 Jul 12;320(3):515-27. doi: 10.1016/s0022-2836(02)00517-x.
Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.
分别在环状质粒底物中对称的FRT和loxP位点上，Flp和Cre介导的重组会产生DNA倒位和缺失。然而，在Flp和Cre的情况下，分别使用由Tn3解离酶形成的resII-resIII突触和噬菌体Mu转座酶形成的LER突触，将三个负超螺旋隔离在重组复合物之外时，反应会以缺失为代价导向倒位。倒位产物是一个三叶结，其独特的拓扑结构由外部解离酶或LER突触赋予。因此，Flp和Cre相对于链切割和交换，为其对称底物赋予了严格的反平行方向。这些结论得到了稀释和竞争试验中拴系平行和反平行天然FRT位点的产物谱的支持。此外，在含有两个对称FRT位点的带切口环状底物中观察到的有利于缺失而非倒位的重组偏向，与基于DNA伙伴反平行突触的蒙特卡罗模拟预测一致。
