Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano - Bicocca, 20126 Milano, Italy.
Nucleic Acids Res. 2019 Apr 23;47(7):3550-3567. doi: 10.1093/nar/gkz038.
Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.
激活检查点蛋白 Tel1 需要 Mre11-Rad50-Xrs2（MRX）复合物，该复合物通过 Tel1 与 Xrs2 之间的直接相互作用将 Tel1 募集到 DNA 双链断裂（DSB）处。然而，MRX 在体外对 Tel1 的激活需要 ATP 结合到 Rad50，这表明 MR 亚复合物在 Tel1 激活中也发挥作用。在这里，我们描述了两种功能分离的等位基因 mre11-S499P 和 rad50-A78T，我们证明它们专门影响 Tel1 激活，而不损害 MRX 在 DSB 修复中的功能。Mre11-S499P 和 Rad50-A78T 都减少了 Tel1-MRX 相互作用，导致 DSB 处 Tel1 结合不良，继而导致 Tel1 激活丧失。Mre11-S499P 变体减少了 Mre11-Rad50 相互作用，这表明 MR 复合物的形成在 Tel1 激活中起着重要作用。分子动力学模拟表明，与 ATP 结合的野生型 MR 亚复合物保持紧密的“关闭”构象，而 ADP 的存在导致 Rad50 二聚体的不稳定性和 Mre11-Rad50 相互作用的不稳定，这两个事件都是 MR 构象向开放状态转变所必需的。相比之下，即使 Rad50 结合了 ATP，MRA78T 也会进行复合物的打开，这表明 MRA78T 导致的 Tel1 激活缺陷是由于结合态的不稳定。
