Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase

Cryo-EM structures and functional analyses of wild-type and constitutively active Mec1-Ddc2 complexes reveal the basis of Mec1 kinase activation and how Dpb11 stimulates Mec1 activity. In response to DNA damage or replication fork stalling, the basal activity of Mec1(ATR) is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of 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 determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 angstrom and the wild type at 3.8 angstrom, 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.

Automated summary

A mutation in Mec1 leading to constitutive activity was identified, and structural analysis revealed the molecular basis for Mec1 activation and key regulatory mechanisms through conformational changes.