Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA.
Department of Biological Sciences, University of Texas at Dallas, Richardson, TX 75080, USA.
Nucleic Acids Res. 2020 May 7;48(8):4371-4381. doi: 10.1093/nar/gkaa153.
In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131-151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.
在 Cre 位点特异性重组中，突触中间体是一种包含一对 loxP DNA 靶位点的重组酶四聚体。该酶系统的链交换机制通过 Holliday 结（HJ）中间体进行；然而，突触中 DNA 片段的几何形状仍然存在很大争议。特别是，所有晶体结构都与非手性、平面 Holliday 结（HJ）结构一致，而基于 Cre 介导的质粒 DNA 扭结的拓扑测定则与右手手性结一致。我们使用涉及紧密间隔（131-151 bp）loxP 位点的环闭合动力学来研究最长寿命的闭环 DNA 中间体的水相构象。将实验环闭合概率 J 的 site-spacing 依赖性拟合到 DNA 环化的统计力学理论提供了大量 HJ 扭曲的证据，这与 Cre-loxP 晶体结构和其他 int-superfamily 重组酶的正方形平面中间体几何形状形成鲜明对比。对 HJ 异构化缺陷 Cre 突变体的 J 测量表明，野生型复合物的表观几何形状与右手和非手性结构的时间平均一致。我们的方法将晶体结构提供的静态图像与溶液中大分子的自然动力学联系起来，从而推进了对大核蛋白结构及其机制的更全面的动态分析。
