Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.
Section of Structural Biology, Department of Infectious Disease, Imperial College London, South Kensington, London, UK.
Nat Struct Mol Biol. 2021 Jan;28(1):50-61. doi: 10.1038/s41594-020-00522-0. Epub 2020 Nov 9.
In response to DNA damage or replication fork stalling, the basal activity of Mec1 is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1 dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.
在应对 DNA 损伤或复制叉停滞时，Mec1 的基础活性以细胞周期依赖性的方式被刺激，导致细胞周期停滞和促进 DNA 修复。Mec1 功能障碍会导致酵母细胞死亡，并导致哺乳动物中的染色体不稳定性和胚胎致死性。因此，ATR 是同源重组缺陷型癌症中癌症治疗的主要靶点。在这里，我们鉴定了 Mec1 中的一个单一突变，该突变在 ATR 中保守，导致组成性活性。使用冷冻电子显微镜，我们在 2.8Å 和 3.8Å 下确定了这种组成性活性形式（Mec1(F2244L)-Ddc2）与 Mg-AMP-PNP 复合物的结构，均为野生型。这些结构为 Mec1-Ddc2 提供了一个近乎完整的原子模型，并揭示了低基础活性和激活所需的构象变化的分子基础。结合生化和遗传数据，我们发现了关键的调节区域，并提出了 Mec1 激活机制。
