Figure S1. Sequence alignments of ATRIP and ATR TopBP1 interacting regions.

Transcription

1 A 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 B VPFRLTH NMVNGMGP MGTEGL FRRACEV TMRLMRDQR EPLMS VLKTFLH DPLVEWSKPVK 2570 ATM 2906 VPFRLTR DIVDGMG ITGVEGV FRRCCEK TMEVMRNSQ ETLLTIV EVLLY DPLFDWTMNPL 2965 DNA-PKcs 3959 MPFRLTR QFINLML PMKETGL MYSIMVHA LRAFRSDPGL LTNT MDVFVKE PSFDWKNFEQ 4018 mtor 2375 IPFRLTR MLTNAMEV TGLDGN YRITCHTV MEVLREHK DSVMA VLEAFVY DPLLNWRLMDT GH SKAPLNE TG EVVN EKAK 2589 ATM 2966 KALYLQQRP --EDETELHP TLNAD D QECKRNLSDIDQSFN 3003 DNA-PKcs 4019 KMLKKGGSWIQEINVA EKNWYPRQKICYAKRKLAG -----ANPAVITCDELLLGHEKAPA 4073 mtor 2435 NTKGNKRSRTRTDSYSAGQ SV EILDGVELGEPAHKKTGTT VPESIHSFIGDGLVKPEALN 2494 * T HVLDIEQ RLQGVI KTRNRVTGLP LSIEG HVHYLI QEATDENL LCQMYL GWTPYM 2644 ATM 3004 KVAERVL MRLQEKL KG--VEEGTV LSVG GQVNLLI QQAIDPK NLSRLFP GWKAWV 3056 DNA-PKcs 4074 F RDYVAV ARGSKDHNI RAQEPESG LSEE TQVKCLM DQATDPNI LGRTWE GWEPWM 4128 mtor 2495 KKAIQII NRVRDKL TGRDFSHDDT LDVPT QVELLI KQATSHE NLCQCYI GWCPFW 2549 Figure S1. Sequence alignments of IP and TopBP1 interacting regions. A) Alignment of the minimal TopBP1 interacting region of human IP and IP homologs of the indicated species. The solid line indicates the position of the IP-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 PRD and the ATM PRD are predicted to contain an -helix 30 amino acids long. The asterisk denotes the location of lysine 2589.

2 A WT VNE2584AAA KAKT2587AAAA HVL2591AAA TopBP1: DIE2594AAA 32 P- Autorad CB B K2 K5 WB K6 IP TopBP1: Autorad CB WB 32 P- IP Figure S2. Kinase assays of PIKK regulatory domain mutants. (A) 293T cells were transfected with IP and wild-type (wt) or containing the indicated alanine substitution mutations. -IP complexes were isolated and incubated with 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- IP and anti- antibodies (WB). (B) 293T cells were transfected with IP and 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 mutants and wild-type is the HVL2591AAA mutant, which consistently demonstrated defective TopBP1-stimulated activity. The increased basal kinase activity exhibited by VNE2584AAA was not reproducible.

3 Flag- WT Myc- WT Flag- K2589E Myc- K2589E Input: Flag Input: Myc IP: Flag WB: Myc IP: Flag WB: Flag IP: Myc WB: Flag IP: Myc WB: Myc Figure S3. K2589E retains the ability to form homo-oligomeric complexes. 293T cells were transfected with the indicated epitope-tagged versions of. 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.

4 WT M1 M2 M3 - IR: DNA-PKcs DNA-PKcs * * DNA-PKcs ps2056 Flag 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.

5 WT C24 C32 ATM PRD+FATC ATM FATC KD Mock IP Autorad CB WB Figure S5. The FATC domain is essential for basal kinase activity. Flag- proteins were isolated from 293T cells transfected with vectors encoding wildtype IP and wild-type (WT), lacking the C-terminal 24 (!C24) or 32 (!C32) amino acids, containing the ATM FATC domain (ATM FATC), containing the ATM PIKK regulatory domain and FATC domains (ATM PRD+FATC), kinase-dead (KD), or mock-transfected (-). Complexes were incubated with 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- antibody (WB).

6 flox/- + K2589E + AdCre flox/- + WT + AdCre NeoExon2 Flox Exon2 Figure S6. Genotype of colonies from flox/- cell lines. Colonies of Cre-recombinase (Ad-Cre) infected flox/- cell lines complemented with K2589E cdna or wild-type (WT) cdna were genotyped using PCR as previously described (Cortez et al., 2001). A representative sample from 60 colonies is shown.