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.