Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase

Abstract

In response to DNA damage or replication fork stalling, the basal activity of Saccharomyces cerevisiae Mec1 ATR is stimulated in a cell cycle dependent manner, leading to cell cycle arrest and promoting DNA repair. Mec1 ATR 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 determined 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 2+ -AMP-PNP. These structures yield near complete atomic models 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.

The activation mechanisms of master kinases in the DNA damage response

“…This effect induces an enclosed nucleotide-binding pocket that enhances ATP binding and hydrolysis (Fig. 3) (Tannous et al, 2021). This activation model is similar to another previous model, in which the potential charge-charge interaction between the AAD of Dpb11 and PRD is capable of destabilizing the interaction between PRD-I and the activation loop to eliminate the PRD-I block (Wang et al, 2017).…”

“…The Mec1-Ddc2 structure forms a similar butterfly shape as seen in ATM/Tel1, forming a dimer of a heterodimer with two-fold rotational symmetry in a head-to-head manner (Fig. 1F) (Tannous et al, 2021;Wang et al, 2017). The cryo-EM reconstructions of Mec1-Ddc2 have revealed that the activation loop attaches to the PRD insert (PRD-I), which seems to be key to inhibiting substrate access (Tannous et al, 2021;Wang et al, 2017).…”

“…1F) (Tannous et al, 2021;Wang et al, 2017). The cryo-EM reconstructions of Mec1-Ddc2 have revealed that the activation loop attaches to the PRD insert (PRD-I), which seems to be key to inhibiting substrate access (Tannous et al, 2021;Wang et al, 2017). This conformation pushes the activation loop far away from the ATP gamma-phosphate for optimal hydrolysis (Fig.…”

“…The structure of an Mg 2+ -AMP-PNP-bound Mec1 (F2244L)-Ddc2 mutant mimics Mec1 in its pre-catalytic state, indicating that PRD-I must move away from the nucleotide-binding pocket to facilitate substrate access and Mec1 activation (Tannous et al, 2021). The disruption between PRD-I and the activation loop allows for the C-terminal of FAT (C-FAT) to move in a clockwise direction relative to the kinase C-lobe and N-lobe.…”

ATR activation is regulated by dimerization of ATR activating proteins

“…Human and yeast ATR-ATRIP complexes form a dimeric butterfly-like structure similar to ataxia telangiectasia mutated (ATM) with two ATR molecules and two ATRIP molecules in a single complex ( 19 , 20 , 21 , 22 , 23 ), and dimerization of the complex is required for function ( 24 ). ATM is activated by the MRE11-RAD50-NBS1 (MRN) complex ( 25 , 26 ), and like ATM, MRN is a dimer with two MRE11, RAD50, and NBS1 molecules in a single complex ( 27 ).…”