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We thank A.V. Failla of the Microscope Core Facility,
K. Hartmann of the Mouse Pathology Core Facility of the University
Medical Center Hamburg-Eppendorf, and V. Vovk of the Lviv
National Medical University for their support. This study was
supported in part by the German Research Society (KFO 306, FU
742/4-1; SFB 841, INST 152/621-1; SFB 877, INST 257/433-1; SFB
1192, INST 152/692-1, INST 152/686-1), the Stiftung für
Pathobiochemie und Molekulare Diagnostik of the German Society
for Clinical Chemistry and Laboratory Medicine, the FoRUM-program of the Ruhr-University Bochum (F505-2006),
Hjärt Lungfonden (20110500), Vetenskapsrådet (K2013-65X-
21462-04-5), and the European Research Council (ERC-StG-2012-
311575_F-12, PIIF-GA-2013-628264). M.H. acknowledges generous
support by Ardea Biosciences, Inc. The data are contained in
the manuscript and the supplementary materials.
Materials and Methods
Figs. S1 to S9
Movies S1 and S2
20 March 2017; resubmitted 10 July 2017
Accepted 11 October 2017
3.9 Å structure of the yeast Mec1-Ddc2
complex, a homolog of human
Xuejuan Wang,1,2 Tingting Ran,3 Xuan Zhang,1 Jiyu Xin,1 Zhihui Zhang,1
Tengwei Wu,1 Weiwu Wang,3 Gang Cai1,2†
The ataxia telangiectasia–mutated and Rad3-related (ATR) kinase is a master regulator of
DNA damage response and replication stress in humans, but the mechanism of its activation
remains unclear. ATR acts together with its partner ATRIP. Using cryo–electron microscopy, we
determined the structure of intact Mec1-Ddc2 (the yeast homolog of ATR-ATRIP), which is poised
for catalysis, at a resolution of 3.9 angstroms. Mec1-Ddc2 forms a dimer of heterodimers through
the PRD and FATdomains of Mec1 and the coiled-coil domain of Ddc2. The PRD and Bridge
domains in Mec1 constitute critical regulatory sites. The activation loop of Mec1 is inhibited by the
PRD, revealing an allosteric mechanism of kinase activation. Our study clarifies the architecture of
ATR-ATRIP and provides a structural framework for the understanding of ATR regulation.
Ataxia telangiectasia–mutated (ATM) and ATM-Rad3–related (ATR) are master regu- lators of the DNA damage response and are highly conserved among eukaryotes. The ATR kinase is essential for the maintenance
of genomic integrity, which is activated by DNA
double-strand breaks (DSBs) as well as various
types of DNA replication problems (1). ATR forms
a stable complex with ATRIP (ATR-interacting
protein), which regulates the localization of ATR
and is essential for ATR signaling (1–3). Muta-
tions in ATR are associated with Seckel syndrome,
a clinically distinct disorder characterized by
proportionate growth retardation and severe
microcephaly (4). Given the central role of ATR
in genome integrity and human disease, it is es-
sential to understand the mechanism of its reg-
ulation. Molecular and structural insights into
ATR are critical to facilitate the design of thera-
peutic agents (5). A negative-stain structure of
Saccharomyces cerevisiae Mec1-Ddc2 (homolog
of human ATR-ATRIP) was recently reported
(6). However, the resolution obtained by electron
microscopy in that study was low (22.5 Å). Here,
we report the cryo–electron microscopy (cryo-
EM) structure of the Mec1-Ddc2 complex at 3.9 Å
resolution (table S1).
The endogenously purified Mec1-Ddc2 com-
plex has a 1:1 stoichiometry, which is consistent
with previous biochemical and functional studies
(fig. S1) (2, 7). The preparation displays basal
kinase activity, which could be stimulated by
incubation with the endogenous Mec1 activator
Dpb11, the homolog of human TopBP1 (fig. S2).
Each Mec1-Ddc2 complex contains two copies of
Mec1 and two copies of Ddc2, such that the com-
plex has a butterfly-like dimeric architecture with
a two-fold rotational (C2) symmetry (Fig. 1, A and
B, and figs. S3 and S4). The dimeric architecture
is similar to that of the Tel1 (homolog of human
ATM) homodimer (8) but is distinct from that of
the m TOR complex (9, 10) and Tor-Lst8 (11) (figs.
S11 to S15). Human ATR can be autophosphoryl-
ated at Thr1989 (12, 13), which is neither close to
the Mec1 kinase domain nor in proximity to the
dimer interface (Fig. 1C). Thus, Thr1989 seems un-
able to access either active site in the Mec1 dimer
without substantial conformational changes.
Mec1 contains a canonical two-lobe kinase
domain (KD) spanning about 400 C-terminal
residues, with three characteristic insertions:
FATC (∼30 residues), LBE (∼40 residues), and
PRD (∼20 residues). The elements crucial for
catalysis are ordered in the structure, including the activation loop, catalytic loop, P-loop,
and FATC (Fig. 1, C and D). Immediately preceding the KD is an array of helical repeats constituting the FAT domain (fig. S5), which extends
toward the N-terminal a-solenoid. The Mec1
N terminus is highly flexible, with more than
200 amino acids (residues 1 to 235) invisible in
the structure. Consistently, no cross-linking signals
were detected within this region (14). Similarly,
the N terminus of the m TOR is also invisible in
the 4.4 Å cryo-EM reconstruction (10).
The Ddc2 density is well defined; most of the
side chains are discernible (fig. S9). Ddc2 has a
sinuous superhelical structure containing 26
helices (13 pairs) of HEAT repeats. Two long
inter-HEAT repeat loops mediate multiple interactions with Mec1 (fig. S10). One loop (residues
338 to 358) abuts the FAT domain (Tyr1573 and
Asn1596) and the upper spiral (Lys1110); the other
one (residues 463 to 535) runs over peripheral
parts of the central hollow region and intertwines with a long inter-HEAT repeat loop of
Mec1 (residues 473 to 516). Interestingly, the reported DNA binding region (Lys177-Lys178-Arg179-
Lys180) in Ddc2 (15) is in close proximity to the
interface with the inter-HEAT repeat of Mec1
There are three major dimer interfaces in
the Mec1-Ddc2 complex (Fig. 2A). The Mec1
PRD [phosphatidylinositol 3-kinase–related kinase (PIKK) regulatory domain] constitutes an
important Mec1-Mec1 dimer interface that critically regulates the kinase activity of PIKKs (16).
The PRD loop interacts with both the ka9b helix
and the TRD3 region of another monomer by
putative hydrogen bonds and electrostatic interactions (Fig. 2B). The TRD2-TRD2 dimer is a
conserved interface shared by Tel1 (8) and Mec1
(Fig. 2C). Several polar or charged residues at
the TRD2 and TRD3 regions are responsible
for making the contacts. The Ddc2-Ddc2 dimer
is formed by the coiled-coil (CC) domain, which
1206 1 DECEMBER 2017 • VOL 358 ISSUE 6367 sciencemag.org SCIENCE
1Hefei National Laboratory for Physical Sciences at
Microscale and School of Life Sciences, University of Science
and Technology of China, Hefei 230027, China. 2CAS Center
for Excellence in Molecular Cell Science, Chinese Academy
of Sciences, Hefei 230026, China. 3Key Laboratory of
Agricultural and Environmental Microbiology, Ministry of
Agriculture, College of Life Sciences, Nanjing Agricultural
University, Nanjing 210095, China.
*These authors contributed equally to this work.
†Corresponding author. Email: firstname.lastname@example.org