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 Mec1ATR is stimulated in a cell cycle dependent manner, leading to cell cycle arrest and promoting 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 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 Mg2+-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.