Abstract
DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50-Nbs1) protein complex through a poorly understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning of the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays.
Highlights
Ataxia-telangiectasia mutated (ATM) is a large protein kinase with a central role in the cellular response to DNA double-strand breaks (DSBs) and related genotoxic stress[1].Mutations in ATM are responsible for Ataxia-telangiectasia (AT), which is a rare, autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, sensitivity to radiation and cancer susceptibility[2].ATM, which belongs to the phosphatidylinositol 3 kinase-like protein kinase (PIKK)family[2], is essential for the sensing of DSBs during the cell cycle
ATM phosphorylates a wide range of downstream effector proteins, such as p53, Chk[2], Brca[2], and H2A.X, leading to the activation of cell cycle checkpoints and homology-directed repair (HDR)[4,5,6,7,8,9]
Fibroblasts from mice harboring an Nbs[1] C-terminal deletion (Nbs1∆C/∆C) do not display overt sensitivity to ionizing radiation (IR), they exhibit defective intra-S phase checkpoint activation, apoptosis induction, and phosphorylation of a subset of ATM targets[30]. These results suggest that the ATM-Nbs[1] interaction is not strictly required to initiate ATM activation, but it is likely necessary to stabilize activated ATM at sites of DNA damage and is critical for certain ATM-mediated checkpoint functions
Summary
Ataxia-telangiectasia mutated (ATM) is a large protein kinase with a central role in the cellular response to DNA double-strand breaks (DSBs) and related genotoxic stress[1].Mutations in ATM are responsible for Ataxia-telangiectasia (AT), which is a rare, autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, sensitivity to radiation and cancer susceptibility[2].ATM, which belongs to the phosphatidylinositol 3 kinase-like protein kinase (PIKK)family[2], is essential for the sensing of DSBs during the cell cycle. Ataxia-telangiectasia mutated (ATM) is a large protein kinase with a central role in the cellular response to DNA double-strand breaks (DSBs) and related genotoxic stress[1]. Mutations in ATM are responsible for Ataxia-telangiectasia (AT), which is a rare, autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, sensitivity to radiation and cancer susceptibility[2]. Family[2], is essential for the sensing of DSBs during the cell cycle. It functions in association with the MRN protein complex consisting of the Mre[11], Rad[50] and Nbs[1] proteins[3]. MRN contributes to the localization of ATM to DSBs, and together with double-stranded DNA (dsDNA), it activates ATM as a protein kinase. Mutations in all three members of the MRN complex cause disorders that are phenotypically similar to AT10–12
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