Figure S1. Sequence alignments of ATRIP and ATR TopBP1...
Transcript of Figure S1. Sequence alignments of ATRIP and ATR TopBP1...
ATR 2511 VPFRLTHNMVNGMGPMGTEGLFRRACEVTMRLMRDQREPLMSVLKTFLHDPLVEWSKPVK 2570
ATM 2906 VPFRLTRDIVDGMGITGVEGVFRRCCEKTMEVMRNSQETLLTIVEVLLYDPLFDWTMNPL 2965
DNA-PKcs 3959 MPFRLTRQFINLMLPMKETGLMYSIMVHALRAFRSDPGLLTNTMDVFVKEPSFDWKNFEQ 4018
mTOR 2375 IPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMAVLEAFVYDPLLNWRLMDT 2424
ATR 2571 GH----------SKAPLNETGEVVN------------- ------------------ EKAK 2589
ATM 2966 KALYLQQRP --EDETELHPTLNADD------------- -------QECKRNLSDIDQSFN 3003
DNA-PKcs 4019 KMLKKGGSWIQEINVA EKNWYPRQKICYAKRKLAG -----ANPAVITCDELLLGHEKAPA 4073
mTOR 2435 NTKGNKRSRTRTDSYSAGQ SVEILDGVELGEPAHKKTGTT VPESIHSFIGDGLVKPEALN 2494
ATR 2590 THVLDIEQRLQGVIKTRNRVTGLPLSIEGHVHYLIQEATDENLLCQMYLGWTPYM 2644
ATM 3004 KVAERVLMRLQEKLKG--VEEGTVLSVGGQVNLLIQQAIDPKNLSRLFPGWKAWV 3056
DNA-PKcs 4074 FRDYVAVARGSKDHNIRAQEPESGLSEETQVKCLMDQATDPNILGRTWEGWEPWM 4128
mTOR 2495 KKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHENLCQCYIGWCPFW 2549
A
B
*
H. sapiens 204 TKLQTS--ERANKLAAPSVSH VSPRKNPSVVIKPEACS-PQFGKTSFPTKESFSANMS LP 259
B. taurus 201 TKLQSS--ERANKLAVPTVSH VSPRKSPSVVIKPEACS-PQFGKPSFPTKESFSANKS LP 257
M. musculus 204 TKSQSN--GRTNKPAAPSVSH VSPRKGSSVVLKSEACS-PHVGKTTFPTKESFSANTP LF 259
R. norvegicus 204 TKLQSN--ERTNKLTIPSVSQ VSPRKGSSVVVKSEACS-PHVGKTTFPTKESFSANTP LF 259
X. laevis 233 TKLQNC--ERSNRTSVP---VVSPKKSPSKGLKSEACSSPLPGRSSFPTKESFCSDMN LR 287
D. rerio 171 TQAQSDREKELS RKVQSLQSE LHFKEAEMNEMRGKLQSVERGGKQSG-TPARQAVKSP LS 229
H. sapiens 260 HPCQTESGYKPLVGREDSK --PHSLRGDSIKQ ---EEAQKSFVDSWRQRSNTQ GSILINL 315
B. taurus 258 QPCQTEPAYKSPMSREVAENKVHSLGGGPIKQ ---EEPQKSLLDSWR --SNNQGSILINL 312
M. musculus 260 HPCQTEAGHRFLVGQEVSDNKNHSLGGSLMKQ ---DVQQRILADGWMQRKDAQ GSILINL 317
R. norvegi cus 260 HPCQTEAGHKFLVGQEVSDNKAHVGG -NLLKQ---DVQQKILTDSWVQRKNTQ GSILINL 316
X. laevis 288 TPPLISAQIGPRTPVISKEPEALPMSSKTFSS ----------- FFYAQRKNSQ GSLLLNA 336
D. rerio 230 RTFMTKENFSAEISKRTSPVKPGPSSDNAALSRCLLPEEQRSQQCIASDLQKE GCVLLKL 289
H. sapiens 316 LLKQPLIPGSSLSLCHLLSSSSESPAGTPLQPP 348
B. taurus 313 LLKQPLIPGSPLGLCHLLSSCPEAPAGTQFQLP 345
M. musculus 318 LLKQPLVPGSSLGLCHLLSSCPEVPTGTLLQPP 350
R. norvegicus 317 LLKQPLVPGSSLGLCHLLSSGAEVPTGPILQPA 349
X. laevis 337 LMQQPICPG-SLGLCNLLSSSTESLPGSPGRNA 369
D. rerio 290 LLQQPLDP-SVLGLCHLLSISPDALPNMLTQHG 322
Figure S1. Sequence alignments of ATRIP and ATR TopBP1 interacting regions.
A) Alignment of the minimal TopBP1 interacting region of human ATRIP and ATRIP
homologs of the indicated species. The solid line indicates the position of the ATRIP-top
mutation. B) Alignment of the C-terminus of human PIKK family members. The solid
line denotes the C-terminal end of the phosphoinositide 3-kinase-related kinase domain.
The dotted line overlies the highly conserved FATC (FRAP, ATM, TRRAP C-terminal)
domain. The PIKK Regulatory Domain (PRD) is between the kinase domain and the
FATC domain. The ATR PRD and the ATM PRD are predicted to contain an -helix 30
amino acids long. The asterisk denotes the location of ATR lysine 2589.
WT
VNE2
584A
AAKA
KT25
87AA
AA
DIE2
594A
AA
HVL2
591A
AA
A
B
ATRATRIP
32P-MCM2
MCM2Autorad
CB
TopBP1: - + - + - + - + - +
ATRATRIP
32P-MCM2
MCM2
K2 K5 K6
CB
WB
TopBP1: - + - + - +
Autorad
WB
Figure S2. Kinase assays of ATR PIKK regulatory domain mutants.
(A) 293T cells were transfected with ATRIP and ATR wild-type (wt) or ATR containing
the indicated alanine substitution mutations. ATR-ATRIP complexes were isolated and
incubated with MCM2 substrate, -32
P-ATP, and recombinant TopBP1 AAD where
indicated. Kinase reactions were separated by SDS-PAGE, stained with coomassie blue
(CB) and exposed to film (autorad). A duplicate gel was blotted and probed with anti-
ATRIP and anti-ATR antibodies (WB). (B) 293T cells were transfected with ATRIP and
ATR containing the following lysine to arginine mutations: K2 (K2568R, K2604R), K5
(K2 + K2571R, K2587R, K2589R), K6 (K5 + K2574R). Kinase reactions were
performed as in (A). The only reproducible difference observed in kinase activity
between these ATR mutants and wild-type ATR is the ATR HVL2591AAA mutant,
which consistently demonstrated defective TopBP1-stimulated activity. The increased
basal kinase activity exhibited by ATR VNE2584AAA was not reproducible.
IP: MycWB: Flag
Input: Flag
IP: FlagWB: Flag
IP: FlagWB: Myc
Input: Myc
IP: MycWB: Myc
+-+--++----+---+Flag-ATR WT
Myc-ATR WTFlag-ATR K2589EMyc-ATR K2589E
Figure S3. ATR K2589E retains the ability to form homo-oligomeric complexes.
293T cells were transfected with the indicated epitope-tagged versions of ATR. Cellular
lysate were pre-cleared with mouse IgG antibody and protein-G beads and then incubated
with anti-myc antibodies and protein-G beads or anti-flag antibody beads. After washing
the beads, bound proteins were eluted, processed by SDS-PAGE, and blotted with the
antibodies against the indicated epitope tag. Input indicates 10% of the lysate used in the
immunoprecipitation reactions. IP= immunoprecipitate, WB= Western blot.
-M1 M2 M3WT
DNA-PKcs pS2056
IR: - + - + - + - + +DNA-PKcs
*
*
Flag*
DNA-PKcs
Figure S4. The DNA-PKcs PIKK regulatory domain is required for DNA-PKcs
autophosphorylation.
V3 DNA-PKcs-defective CHO cells were transfected with wild-type (WT) flag-DNA-
PKcs, flag-DNA-PKcs PRD mutants (M1:K4043E/K4048E/R4049E/K4050E,
M2:D4062K/E4063K/E4069K, M3:K4075E/R4082E/R4085E/R4090E), or mock (-)
transfected. Cells were treated with 10 Gy ionizing radiation (IR) or mock treated and
harvested 1 hr later. Cell lysates were analyzed by immunoblotting using anti-pS2056
DNA-PKcs and anti-flag antibodies. Asterisks indicate non-specific cross-reactions
bands.
WB
∆C24
∆C32
KD Mock
ATM FA
TC
ATR
ATRIP
Autorad
WT
CB
ATM PRD+F
ATC
MCM2
MCM2
ATR
Figure S5. The ATR FATC domain is essential for basal ATR kinase activity.
Flag-ATR proteins were isolated from 293T cells transfected with vectors encoding wild-
type ATRIP and wild-type (WT) ATR, ATR lacking the C-terminal 24 (!C24) or 32
(!C32) amino acids, ATR containing the ATM FATC domain (ATM FATC), ATR
containing the ATM PIKK regulatory domain and FATC domains (ATM PRD+FATC),
ATR kinase-dead (KD), or mock-transfected (-). Complexes were incubated with MCM2
substrate and -32
P-ATP. Kinase reactions were separated by SDS-PAGE, stained with
coomassie blue (CB) and exposed to film (autorad). A duplicate gel was blotted and
probed with an anti-ATR antibody (WB).
NeoExon2Flox
∆Exon2
ATRflox/- +ATR K2589E+ AdCre
ATRflox/- + ATR WT+ AdCre
Figure S6. Genotype of colonies from ATRflox/-
cell lines.
Colonies of Cre-recombinase (Ad-Cre) infected ATRflox/-
cell lines complemented with
ATR K2589E cDNA or ATR wild-type (WT) cDNA were genotyped using PCR as
previously described (Cortez et al., 2001). A representative sample from 60 colonies is
shown.