LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex.
LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex.
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DOI：
10.7554/elife.69579
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发表时间：
2022-01-27
期刊：
影响因子：
7.7
通讯作者：
Latham MP
中科院分区：
文献类型：
--
作者：
Canny MD;Latham MP
Mre11-Rad50-Nbs1蛋白复合体是DNA双链断裂的第一反应因子之一。研究表明，进化保守的Mre11-Rad50(MR)核心复合体的催化活性依赖于依赖于ATP的全球构象变化，当ATP结合时，这种变化使大分子从缺乏ATP的开放的、延伸的结构转变为封闭的球状结构。我们之前已经通过发光共振能量转移(LRET)实验确定了另一种‘部分开放’构象。在这里，LRET和分子对接程序HADDOCK被用来进一步研究这种部分开放的状态，并鉴定了MR在溶液中的三种构象：封闭的、部分开放的和开放的，这是除了扩展的载脂蛋白构象之外的三种构象。破坏每个构象内特定Mre11-Rad50相互作用的突变体被用于各种DNA底物上的核酸酶活性分析，以帮助将这三种状态置于功能角度。磁共振结合DNA的LRET数据表明，当核酸酶底物结合时，这三种构象也存在。这些模型进一步得到了小角X射线散射数据的支持，证实了溶液中存在多个态。总之，这些数据表明了沿着DNA的MR复合体的核酸酶活性的机制。
The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double-strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using luminescence resonance energy transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with small-angle X-ray scattering data, which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.