Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET
Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET
复制标题
DOI：
10.1073/pnas.1309816110
复制
发表时间：
2013-11-19
影响因子：
11.1
通讯作者：
Paull, Tanya T.
中科院分区：
文献类型：
--
作者：
Cannon, Brian;Kuhnlein, Jeffrey;Paull, Tanya T.
Mre 11/Rad 50/Nbs 1（MRN）复合物在双链断裂时启动和协调DNA修复和信号传导事件。MRN和DNA末端之间的相互作用对于DNA加工酶的募集、末端束缚和ATM蛋白激酶的活化至关重要。在这里，我们使用单分子FRET可视化MRN与双链体DNA分子的结合，并发现MRN在双链体末端解旋15-20个碱基对，在ATP依赖性反应中保持分支结构开放数分钟。一个Rad 50催化结构域突变体，这是特别缺乏这种ATP依赖性开放是受损的DNA末端切除在体外和切除依赖性修复的人细胞中的断裂，证明了MRN产生的单链DNA断裂修复的重要性。
The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.