TY - JOUR
T1 - Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase
AU - Tannous, Elias A.
AU - Yates, Luke A.
AU - Zhang, Xiaodong
AU - Burgers, Peter M.
N1 - Funding Information:
We thank Burgers laboratory members C. Stith and B. Yoder for strains construction, and J. Haber (Brandeis University) for plasmids. We thank A. Nan (Francis Crick Institute), K. Cunnea (eBIC) and Y. Song (eBIC) for their support with cryo-EM data acquisition and Zhang laboratory members R. Ayala, for help with initial screening, and R. Williams, for discussions. Initial cryo-EM screening of samples was carried out at the Imperial College London Center for Structural Biology EM facility. High resolution cryo-EM data were collected at eBIC (proposal no. EM19865). eBIC is funded by the Wellcome Trust, MRC and BBSRC. This work was funded in part by grant no. GM118129 from the National Institutes of Health (to P.M.B.) and the Wellcome Trust grant no. 210658/Z/18/Z (to X.Z.).
Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85095696698&partnerID=8YFLogxK
U2 - 10.1038/s41594-020-00522-0
DO - 10.1038/s41594-020-00522-0
M3 - Article
C2 - 33169019
AN - SCOPUS:85095696698
VL - 28
SP - 50
EP - 61
JO - Nature Structural and Molecular Biology
JF - Nature Structural and Molecular Biology
SN - 1545-9993
IS - 1
ER -