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Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase.

<div class='caption-title'>(a) Simplified model of the yeast DNA replication fork and replication stress response through Mec1-Ddc2 and its activators. The yeast activators shown are the Ddc1Rad9 subunit of the 9-1-1 checkpoint clamp, the replication initiator protein Dbp11TopBP1, and the nuclease-helicase Dna2. These three proteins have unstructured CTDs (Ddc1, Dpb11) or NTD (Dna2), which mediate activation.</div>
<div class='caption-title'>(a) Phosphorylation of Rad53 by Mec1 or Mec1-x mutants with the indicated concentrations of Dpb11 activator. To eliminate contributions by Rad53 kinase itself, the kinase-dead version was used (Rad53-kd, K227A). Example of a full gel is shown in Extended Data Fig. 1a. Uncropped gel images are shown in the Source Data.</div>
<div class='caption-title'>(a) Two orthogonal views of the Mec1-Ddc2 AMP-PNP-bound complex resolved to 3.8 Å, and (b) two orthogonal views of the constitutively active Mec1(F2244L)-Ddc2:AMP-PNP complex resolved to 2.8 Å resolution. Structurally distinct domains are colored; red, kinase; pink, C-terminal-FAT; yellow, Middle-FAT; blue, N-terminal-FAT; navy, bridge; green, C-terminal HEAT; teal, N-terminal HEAT. The Ddc2 subunit is colored purple and the nucleotide colored bright green. The C2 axis is shown. The second Mec1-Ddc2 heterodimer is shown in grey (Mec1’) and dark purple (Ddc2’).</div>
Tannous EA, Yates LA, Zhang X, Burgers PM
OpenAccess logo Nat Struct Mol Biol 28 50-61 (2021)
Related entries: 6z2x, 6z3a

Cited: 12 times
EuropePMC logo PMID: 33169019

Abstract

In response to DNA damage or replication fork stalling, the basal activity of Mec1ATR is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1ATR 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 Mg2+-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.

Articles - 6z2w mentioned but not cited (2)

  1. Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase. Tannous EA, Yates LA, Zhang X, Burgers PM. Nat Struct Mol Biol 28 50-61 (2021)
  2. Novel insights into the mechanism of cell cycle kinases Mec1(ATR) and Tel1(ATM). Tannous EA, Burgers PM. Crit Rev Biochem Mol Biol 56 441-454 (2021)


Reviews citing this publication (4)

  1. Mechanism of activation and the rewired network: New drug design concepts. Nussinov R, Zhang M, Maloney R, Tsai CJ, Yavuz BR, Tuncbag N, Jang H. Med Res Rev 42 770-799 (2022)
  2. Recent Advances in Synergistic Antitumor Effects Exploited from the Inhibition of Ataxia Telangiectasia and RAD3-Related Protein Kinase (ATR). Wang LW, Jiang S, Yuan YH, Duan J, Mao ND, Hui Z, Bai R, Xie T, Ye XY. Molecules 27 2491 (2022)
  3. Structure and function of the apical PIKKs in double-strand break repair. Xu J, Bradley N, He Y. Curr Opin Struct Biol 82 102651 (2023)
  4. Tools for Decoding Ubiquitin Signaling in DNA Repair. Foster B, Attwood M, Gibbs-Seymour I. Front Cell Dev Biol 9 760226 (2021)

Articles citing this publication (6)

  1. DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp. Zheng F, Georgescu RE, Yao NY, O'Donnell ME, Li H. Nat Struct Mol Biol 29 376-385 (2022)
  2. Molecular basis of human ATM kinase inhibition. Stakyte K, Rotheneder M, Lammens K, Bartho JD, Grädler U, Fuchß T, Pehl U, Alt A, van de Logt E, Hopfner KP. Nat Struct Mol Biol 28 789-798 (2021)
  3. ATR activation is regulated by dimerization of ATR activating proteins. Thada V, Cortez D. J Biol Chem 296 100455 (2021)
  4. A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A. Yates LA, Tannous EA, Morgan RM, Burgers PM, Zhang X. Proc Natl Acad Sci U S A 120 e2300150120 (2023)
  5. Structural insights into the activation of ataxia-telangiectasia mutated by oxidative stress. Howes AC, Perisic O, Williams RL. Sci Adv 9 eadi8291 (2023)
  6. Letter Structures of Mec1/ATR kinase endogenously stimulated by different genotoxins. Zhang Q, Wang P, Wu T, Zhang Y, Zheng Z, Zhou S, Qian D, Wang X, Cai G. Cell Discov 8 98 (2022)


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