The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism
The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism
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DOI：
10.1016/s1097-2765(03)00342-3
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发表时间：
2003-09-01
期刊：
影响因子：
16
通讯作者：
Russell, P
中科院分区：
文献类型：
--
作者：
Gaillard, PHL;Noguchi, E;Russell, P
功能研究强烈表明，Mus81-Eme1复合物解决了裂殖酵母中的霍利迪连接（HJ），但在体外，它优先切割复制叉或3 '瓣模型的柔性三向分支结构。在这里，我们报告说，一个缺口HJ是内源性和重组Mus81-Eme1的首选底物。切割特异性地发生在与切口相对的链上，导致结构分解成线性双链体产物。由内源性Mus81-Eme1复合物在完整的HJ上产生的分解切割是准同时的，这表明Mus81-Eme1通过切口和反切口机制分解HJ，其中第二切割的大速率增强来自切口HJ中间体的柔性性质。重组Mus 81-Eme 1在第一次切割时无效。我们还报道了HJ在DNA聚合酶a中积累。缺乏Mus81的突变体，提供了Mus81-Eme1复合物在体内靶向HJ的进一步证据。
Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase a. mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.