Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble
Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble
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DOI：
10.1073/pnas.1820157116
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发表时间：
2019-03-19
影响因子：
11.1
通讯作者：
Cejka, Petr
中科院分区：
文献类型：
--
作者：
Cannavo, Elda;Reginato, Giordano;Cejka, Petr
为了通过同源重组修复 DNA 双链断裂，必须首先切除 5' 末端的 DNA 链以产生 3' 突出端。来自酿酒酵母的 Mre11 是一种 3'→5' 核酸外切酶，负责体内 5' 末端降解。使用质粒长度的 DNA 底物和纯化的重组蛋白，我们发现重组 MRX-Sae2 的组合核酸外切酶和核酸内切酶活性优先降解延伸到 DNA 末端附近的 5' 末端 DNA 链。从机制上讲，Rad50 以 ATP 结合依赖性方式限制 Mre11 核酸外切酶，防止 3' 末端降解。磷酸化的 Sae2 与前面所示的 MRX 核酸内切酶刺激一起，也克服了这种抑制作用，促进了 MRX 的 3'-> 5' 核酸外切酶，这需要 Rad50 进行 ATP 水解。我们的结果支持这样的模型：MRX-Sae2 通过逐步内切 DNA 切口催化 5'-DNA 末端降解，然后对单个 DNA 片段进行外切核酸切 3'-> 5' 降解。该模型解释了 Mre11 的核酸外切酶和核酸内切酶活性如何在 MRX-Sae2 整体中功能性整合以切除 5' 末端 DNA。
To repair DNA double-strand breaks by homologous recombination, the 5'-terminated DNA strands must first be resected to produce 3' overhangs. Mre11 from Saccharomyces cerevisiae is a 3'-> 5' exonuclease that is responsible for 5' end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5'-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3' end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3'-> 5' exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5'-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3'-> 5' degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5'-terminated DNA.