Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase.
Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase.
复制标题
通过 Flp 位点特异性重组酶解析束缚的反平行和平行霍利迪连接。
DOI：
10.1006/jmbi.1999.3472
复制
发表时间：
2000
影响因子：
5.6
通讯作者：
Jayaram,M
中科院分区：
文献类型：
--
作者：
Lee,J;Tribble,G;Jayaram,M
整合酶家族位点特异性重组酶（也称为酪氨酸家族）的成员通过一次交换一对单链来分两步实现重组。第一个交换反应的产物是四向 DNA 连接，即霍利迪中间体。重组复合物从霍利迪形成模式“异构化”到霍利迪分解模式的构象动力学尚不清楚。使用 lambda Int 和大肠杆菌 XerC/XerD 系统进行的实验表明，无蛋白质连接处分支点的链构型决定了蛋白质结合连接处的解析模式。我们通过使用一系列合成霍利迪连接来检查 Flp 系统解析过程中的链偏倚问题，这些合成霍利迪连接在构象上受到局部序列或链束缚的限制。我们没有观察到有利于在未结合的连接点内呈现“交叉”构型的链的强烈分辨率偏差。各种底物环境中反平行连接的分辨率模式揭示了链选择的均等性，或仅适度的差异。另一方面，在系留平行连接的情况下观察到的高度偏差的分辨率可以通过这些底物的DNA臂的蛋白质占据的不等价性和/或在系留末端将切割事件转化为重组体的低效转化来解释。
Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex “isomerizes” from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the “crossed” configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.