Supplementary Text 2-4 Supplementary Tables 5-6 Supplementary Movies Legends 7 Supplementary Figures Model Source Files (8 appended files)

Transcription

1 Supplementary Material Content Page Supplementary Text 2-4 Supplementary Table 5-6 Supplementary Movie Legend 7 Supplementary Figure Model Source File (8 appended file)

2 Supplementary Text Computational model of the repoe to UV We generated a model of the repoe to UV (UV_model.m and command_uv_model.m) baed on our previouly publihed model of the repoe to double trand break (DSB) (Batchelor et al., 28) uing imilar pecie and parameter value. We included new term in the DSB model (DSB_model.m and command_dsb_model.m) to account for the dephophorylation of by (Lu et al., 25). Since -mediated ubiquitination of i the dominant factor governing degradation, we approximated the parameter governing the - mediated dephophorylation of to be one-tenth the value for the parameter governing -mediated degradation. The addition of thee term did not qualitatively alter the ocillatory behavior of in the DSB model (Figure 2C of the main text and (Batchelor et al., 28)). To cotruct the UV model, we eliminated the term in the DSB model decribing the interaction between and the uptream kinae, ATM (Figure 2A-B, main text). We have alo reduced the trength of the ATR/ interaction term to account for the fact that while phophorylation of by ATM affect both activity and tability, phophorylation of by ATR affect only activity (Stommel and Wahl, 24 and main text). Thi reulted in the following et of delay differential equatio decribing the UV repoe: + α wpa in [ [ [ p m[ w[ ATR 2 p α mpi [ [ α [ pi in [ ATR [ ATR ( t τ ) + β ( t τ ) α [ w m [ ATR [ θ ( t) θ ( t t ) α [ ATR r in w mi in + α β [ T m2 p α mpa in [ ATR [ ATR [ [ α [ α [ m + T wpa [ [ 2

3 Parameter value are lited in Supplementary Table. All numerical imulatio were performed in Matlab uing dde23. Value for the production rate of ATR, β 2, and the duration of ATR ignaling, t r, for each modeled UV doe are lited in Supplementary Table 2. + α wpa ATR in Computational model of cro-talk between the ATM and ATR pathway We generated a combined model to explore the poible contributio of cro-talk between the UV/ATR and the gamma/atm pathway (DSB_crotalk_model.m, command_dsb_crotalk_model.m, UV_crotalk_model.m, and command_uv_crotalk_model.m) (Jazayeri et al., 26)(Yajima et al., 29). Thi reulted in the following et of differential equatio: [ [ [ m[ w[ ATM P p p 2 α mpi [ [ α [ pi in ( t τ ) + β ( t τ ) α [ [ θ ( t) θ ( t t ) n [ ATM P [ ATM P + T w m [ [ [ θ ( t) θ ( t t ) α [ ATR r r in w mi α in w β [ m nw p [ ATR + [ ATR nw + T nw w in n [ ATM P [ ATM P + T + T α [ ATM P[ α m2 α [ ATR [ ATM P α [ ATM P mpa [ ATR + [ ATR [ [ α [ α [ m + T wpa [ [ We ued value of α w = 5 h -, T w =.2 C, and n w = 4, a were ued in the previouly publihed model for the repoe to DSB (Batchelor et al., 28). To etimate the amount of cro-talk between the ignaling kinae, we meaured the level of Chk phophorylated on Ser-37 and Chk2 phophorylated on Thr-68, target of ATR and ATM, repectively (Matuoka et al., 2; Zhao and Piwnica-Worm, 2), in repoe to γ or UV irradiation (Figure 2G-H, Main Text). The ratio of maximum phopho-chk(s37) level in repoe to γ compared with it level in repoe to UV wa.35. Therefore, to model the cro-talk of ATR in repoe to γ we 3

4 et β 2,DSB =.35 * β 2,UV. Similarly, the ratio of maximum phopho-chk2(t68) level in repoe to UV compared with it level in repoe to γ wa.8. Therefore, to model the cro-talk of ATM in repoe to UV we et β,uv =.8 * β,dsb. In repoe to DSB, we ued β = C h - (Batchelor et al, 28). Simulation of the model indicated that there wa minimal difference in dynamic between model including or excluding the cro-talk in repoe to either γ or UV (Supplementary Figure 3; compare A to B and C to D). Supplementary Reference Batchelor, E., Mock, C.S., Bhan, I., Loewer, A., and Lahav, G. (28). Recurrent initiation: a mechanim for triggering pule in repoe to DNA damage. Mol Cell 3, Jazayeri, A., Falck, J., Luka, C., Bartek, J., Smith, G.C., Luka, J., and Jackon, S.P. (26). ATM- and cell cycle-dependent regulation of ATR in repoe to DNA doubletrand break. Nat Cell Biol 8, Lu, X., Nannenga, B., and Donehower, L.A. (25). PPMD dephophorylate Chk and and abrogate cell cycle checkpoint. Gene Dev 9, Matuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., and Elledge, S.J. (2). Ataxia telangiectaia-mutated phophorylate Chk2 in vivo and in vitro. Proc Natl Acad Sci U S A 97, Yajima, H., Lee, K.J., Zhang, S., Kobayahi, J., and Chen, B.P. (29). DNA doubletrand break formation upon UV-induced replication tre activate ATM and DNA- PKc kinae. J Mol Biol 385, 8-8. Zhao, H., and Piwnica-Worm, H. (2). ATR-mediated checkpoint pathway regulate phophorylation and activation of human Chk. Mol Cell Biol 2,

5 Parameter Decription Value β p in production rate.9 C h - β p Saturating production rate of h - β m -dependent production rate.9 h - β mi -independent production rate.2 C h - β w production rate.25 h - α mpi -dependent in degradation rate 5 C - h - α pi In degradation rate 2 h - α mpa -dependent degradation rate.4 C - h - α wpa -dependent degradation rate.4 C - h - α m2 ATR -dependent inactivation rate. C - h - α m degradation rate h - α w degradation rate.7 h - α ATR degradation rate 7.5 h - τ m Time delay in production.7 h τ w Time delay in production.25 h T Signal for half-maximal production C n Hill coefficient of production by ATR 4 in Initial in.3 C P53 Initial C Initial.2 C Initial C ATR Initial ATR C Supplementary Table. Parameter and initial conditio of the UV-repoe model. C = imulated unit. 5

6 Simulated UV doe (J/m 2 ) Active ignaling time t r (h) ATR production rate β 2 (C h - ) Supplementary Table 2. ATR production rate, β 2, and ATR ignaling time, t r, for each UV doe modeled. C = imulated unit. 6

7 Supplementary Movie. repoe to DSB. MCF7 cell expreing -Venu (peudocolored green) were treated with 4ng/ml of NCS. Movie duration: 6 hour. Image were taken every 2 minute. Supplementary Movie 2. repoe to UV. MCF7 cell expreing -Venu (peudocolored green) were treated with J/m 2 of UV. Movie duration: 6 hour. Image were taken every 2 minute. 7

8 % of cell 6 4 ng/ml NCS Dominant period (h) % of cell % of cell % of cell % of cell % of cell J/m 2 UV 4 J/m 2 UV 6 J/m 2 UV 8 J/m 2 UV J/m 2 UV Dominant period (h) Supplementary Figure. Pitch analyi indicate that there i no ingle characteritic period of pule in repoe to UV. Pitch analyi of the trace of -Venu in repoe to 4 ng/ml NCS (left column) or to variou doe of UV (right column) wa performed a previouly decribed (Geva-Zatorky et al., 26).

9 A Simulated low doe of γ ATM-P total C Simulated low doe of UV ATR total B Simulated high doe of γ ATM-P total D Simulated high doe of UV ATR total Supplementary Figure 2. Removal of the inhibition of uptream kinae activity by eliminate repeated pule. Simulatio of the dynamic of ATM-P, ATR,,, and. All value for parameter are given in (Batchelor et al., 28) for the γ repoe (A, B) or Supplementary Table for the UV repoe (C, D), with β 2 = C /h. The time of repair wa imulated at h for low doe of γ (A) and UV (C), and 27 h for high doe of γ (B) and UV (D). Note that the γ doe determine the number of pule, while the UV doe affect the duration of a ingle pule.

10 A B Simulated DSB repoe without ATR cro-talk.4 ATM-P.2 ATR total Simulated DSB repoe with ATR cro-talk ATM-P ATR total C D Simulated UV repoe without ATM cro-talk Simulated UV repoe with ATM cro-talk ATM-P ATR total ATM-P ATR total Supplementary Figure 3. Cro-talk between ATM and ATR doe not qualitatively alter the dynamic of the repoe to γ and UV. (A, B) Simulatio of the dynamic of ATM-P, ATR,,, and in the abence (A) or preence (B) of cro-talk from the ATR pathway in repoe to DSB. (C, D) Simulatio of the dynamic of ATM-P, ATR,,, and in the abence (C) or preence (D) of cro-talk from the ATM pathway in repoe to 8 J/m 2 UV. Equatio and parameter value for the imulatio are given in Supplementary Text and Supplementary Table.

11 A Gy γ B 8 J/m 2 UV control +Wm control +Wm Chk2-P(T68) (ATM activity) Chk-P(S37) (ATR activity) Chk2 Chk Tubulin Tubulin Supplementary Figure 4. Wortmannin inhibit ATM and ATR activity. MCF7 cell were irradiated with Gy of γ (A) or 8 J/m 2 of UV (B). One hour after irradiation, DMSO (control) or µm of wortmannin (+Wm) were added to the cell medium. Wetern blot analyi of whole cell lyate wa performed to detect level of phophorylated Chk2(T68) (A) or phophorylated Chk(S37) (B).

12 A 8 J/m 2 UV 2 control 2 +Wm -Venu (AU) B Fraction of cell Venu (AU) 8 4 α β.5 α/β control +Wm Supplementary Figure 5. The repoe to UV i not excitable even at later time following UV damage. (A) Cell expreing -Venu were treated with 8J/m 2 UV. Three hour after damage, medium containing DMSO (control) or wortmannin (+Wm) wa added (dahed line). Repreentative background-ubtracted ingle cell trace of average -Venu inteity are hown for each condition. (B) Hitogram of the ratio of - Venu inteity at 6h after UV to -Venu inteity at time (3h) of DMSO or Wm addition for the experiment hown in (A) (~ cell/condition).