Faculty of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nara City, Nara, 631-8505, Japan.
Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), 6500, Bellinzona, Switzerland.
Nat Commun. 2024 Aug 22;15(1):7221. doi: 10.1038/s41467-024-51493-5.
DNA double-strand breaks (DSBs) must be repaired to ensure cell survival and genomic integrity. In yeast, the Mre11-Rad50-Xrs2 complex (MRX) collaborates with Sae2 to initiate DSB repair. Sae2 stimulates two MRX nuclease activities, endonuclease and 3'-5' exonuclease. However, how Sae2 controls the two nuclease activities remains enigmatic. Using a combined genetic and biochemical approach, we identified a separation-of-function rad50 mutation, rad50-C47, that causes a defect in Sae2-dependent MRX 3'-5' exonuclease activity, but not endonuclease activity. We found that both the endo- and 3'-5' exonuclease activities are essential to release Spo11 from DNA ends, whereas only the endonuclease activity is required for hairpin removal. We also uncovered that MRX-Sae2 endonuclease introduces a cleavage at defined distances from the Spo11-blocked end with gradually decreasing efficiency. Our findings demonstrate that Sae2 stimulates the MRX endo- and exonuclease activities via Rad50 by different mechanisms, ensuring diverse actions of MRX-Sae2 nuclease at DNA ends.
DNA 双链断裂 (DSBs) 必须被修复以确保细胞存活和基因组完整性。在酵母中，Mre11-Rad50-Xrs2 复合物 (MRX) 与 Sae2 合作启动 DSB 修复。Sae2 刺激 MRX 内切核酸酶和 3'-5' 外切核酸酶的两种活性。然而，Sae2 如何控制这两种核酸酶活性仍然是一个谜。我们使用组合的遗传和生化方法，鉴定了一个分离功能的 rad50-C47 突变，该突变导致 Sae2 依赖性 MRX 3'-5' 外切核酸酶活性缺陷，但内切核酸酶活性正常。我们发现，内切核酸酶和 3'-5' 外切核酸酶活性对于从 DNA 末端释放 Spo11 都是必需的，而只有内切核酸酶活性对于发夹去除是必需的。我们还发现，MRX-Sae2 内切核酸酶在 Spo11 阻断的末端以逐渐降低的效率在特定距离处产生切割。我们的研究结果表明，Sae2 通过 Rad50 以不同的机制刺激 MRX 内切核酸酶和外切核酸酶活性，确保了 MRX-Sae2 核酸酶在 DNA 末端的不同作用。
