Deshpande Rajashree A, Lee Ji-Hoon, Paull Tanya T
Howard Hughes Medical Institute, Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Nucleic Acids Res. 2017 May 19;45(9):5255-5268. doi: 10.1093/nar/gkx173.
The Mre11-Rad50-Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound 'closed' state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells.
Mre11-Rad50-Nbs1（Xrs2）（MRN/X）复合物对于DNA双链断裂的修复和信号传导至关重要。MRN/X的催化核心由Mre11核酸酶和Rad50腺苷三磷酸酶（ATP酶）活性位点组成，通过Rad50 ATP酶催化结构域之间的缔合形成二聚体，并在ATP结合后发生广泛的构象变化。这种ATP结合的“封闭”状态促进与DNA的结合、连接DNA末端并激活ATM，但会阻止DNA末端的核酸酶加工，而ATP水解对于Mre11核酸内切酶在受阻DNA末端的活性至关重要。在这里，我们研究了ATP水解的调节以及两个功能活性位点的相互依赖性。我们发现双链DNA以末端依赖性方式刺激hMRN的ATP水解超过20倍。使用催化位点突变体创建仅具有一个功能性ATP酶位点的Rad50二聚体，我们发现两个ATP酶位点都是DNA刺激所必需的。MRN介导的在蛋白质加合物位点对DNA的核酸内切酶切割需要两个位点的ATP水解，对ATM激酶活性的刺激也是如此。这些观察结果表明，Rad50催化头部结构域与两个位点结合的ATP的对称结合对于真核细胞中MRN的功能很重要。
