Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast
Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast
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DOI：
10.1038/emboj.2012.345
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发表时间：
2013-02-06
期刊：
影响因子：
11.4
通讯作者：
Lee, Sang Eun
中科院分区：
文献类型：
--
作者：
Li, Fuyang;Dong, Junachao;Lee, Sang Eun
酿酒酵母Rad1/Rad10复合物是一种多功能、结构特异性的核酸内切酶，可处理紫外线诱导的DNA损伤、重组中间体和DNA链间交联。然而，我们不知道Rad1/Rad10如何识别这些结构上不同的靶分子，也不知道它如何被纳入能够切割不同底物的蛋白质复合物中。在这里，我们确定了Rad1/Rad10复合物、Saw1、Slx4和Msh2/Msh3复合物在3 '尾重组中间体上的组装顺序和层次结构。我们发现Saw1是一种结构特异性的DNA结合蛋白，对三叉臂和3 '瓣DNA具有高亲和力。通过物理相互作用，Saw1促进了Rad1在体内和体外靶向3 ‘尾底物，并在纯化的体外系统中增强了Rad1/Rad10对3 ’尾的切割。我们的结果使我们能够建立一个Rad1/Rad10/Saw1核酸酶复合物组装和重组中3 '尾部去除的模型。
The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3 ' tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3 ' flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3 ' tailed substrates in vivo and in vitro, and enhances 3 ' tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3 ' tail removal in recombination.