Supplemental Information

Supplemental Methods

Protein pulldown with recombinant SUMO

Nuclei of HeLa cells were isolated in buffer H (10 mM HEPES pH7.9, 50 mM NaCl, 0.1 mM EDTA, 0.5 M Sucrose, 1 mM DTT, 20 mM NEM, 0.5% Triton X-100, and protease inhibitors), and subsequently lysed with buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 20 mM NEM, and protease inhibitors). Nuclear pellets were extracted with buffer C (10 mM HEPES pH7.9, 600 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 20 mM NEM, 0.15% NP-40, and protease inhibitors) to generate nuclear extracts. Nuclear extracts were diluted in buffer C without NaCl to bring the final NaCl concentration to 150 mM. Diluted nuclear extracts were mixed with 25 mg GST-1xSUMO2 or GST-4xSUMO2 purified from E. coli at 4oC overnight, and then incubated with 10 ml Glutathione Sepharose 4B beads for 2 hours. After extensive wash, proteins bound to the beads were eluted with 30 ml 1x SDS sample buffer and subjected to Western blotting.

To test whether TopBP1 and MRN interact with SUMO2 directly, we purified GST-TopBP1 from E. coli and recombinant MRN complex from baculovirus-infected insect cells. Purified TopBP1 and MRN were incubated with His-1xSUMO2 or His-4xSUMO2 purified from E. coli. After the incubation, His-tagged SUMO2 and the associated proteins were retrieved with Ni-NTA beads and analyzed by Western.

In vitro SUMOylation assay

Recombinant GST-ATRIP214-328 (WT and 2KR) protein was purified from E. coli using glutathione beads (GE Healthcare Life Sciences). In vitro SUMOylation reactions were conducted using the SUMOylation kit from BostonBiochem and 20 mg GST-ATRIP214-328. The reactions were carried out at 37 °C for 1 hr.

Partial protease digestion

Hela cells transfected with SFB-ATRIPWT or SFB-ATRIP2KR were lysed in RIPA buffer containing 500 mM NaCl. SFB-ATRIP proteins were immunoprecipitated using anti-Flag antibody. The immunoprecipitated SFB-ATRIP was digested with increasing amounts of proteinase K (0, 3, 10, 30 and 100 ng) at room temperature for 3 min. The digestion was terminated by boiling the samples in the SDS sample buffer for 15 min.

The clonogenic assay

Two days after transfection of control or ATRIP 3’ UTR siRNA, HeLa cells stably expressing SFB-ATRIPWT, SFB-ATRIP2KR, or carrying the vector were trypsinized, counted, and plated to 6-well plates at the density of 400 cells/well. The cells were irradiated with different doses of UV 20 hr later. Cells were fixed with methanol and stained by 0.5% crystal violet in 10-12 days.

Flow cytometry and EdU labeling

Cells were trypsinized, washed with 1xPBS, and fixed in 75% ethanol at -20 °C. After fixation, cells were spun down and washed with 1xPBS containing 0.1 mM EDTA, and incubated with propidium iodine (PI) and RNase A at 37 °C for 20 min. Data acquisition was performed on a FACS LSRII apparatus equipped with the FACS Diva software (BD Biosciences). To monitor DNA synthesis, cells were labeled with 10 mM EdU for 30 min and processed using a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay kit according to manufacter’s instructions (Invitrogen).

The S-phase checkpoint assay

Cells were either irradiated with ionizing radiation (IR, 10 Gy) or left untreated. One hour later, cells were labeled with 10 µM EdU for 30 min and processed using a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay kit according to manufacter’s instructions (Invitrogen). Data acquisition was performed on a FACS LSRII apparatus equipped with the FACS Diva software (BD Biosciences). Further analysis and quantification of EdU-positive cells in untreated and IR-treated cell populations were done with Kaluza software (Beckman Coulter). The gating for highly replicating cells was set according to the top 80% of the EdU-positive, untreated ATRIP2KR-expressing cells, and was applied to all samples.

Live cell imaging and focal laser microirradiation

Live cell imaging combined with laser microirradiation was carried out as described previously with modifications (Uematsu et al., 2007). Fluorescence was monitored by using an Axiovert 200M microscope (Carl Zeiss, Inc.), with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss, Inc.). A 365-nm pulsed nitrogen laser (Spectra-Physics) was directly coupled to the epifluorescence path of the microscope and used to generate DSBs in a defined area of the nucleus. For quantitative analyses, standardized micro-irradiation conditions (minimal laser output of 80% for 5 pulses) were used to generate the same amount of DNA damage in each experiment. Time-lapse images were taken by an AxioCamHRm camera and the fluorescence intensities of microirradiated and non-irradiated areas within the cell nucleus were determined using the Axiovison Software, version 4.8 (Carl Zeiss, Inc.). To eliminate the influence of nuclear background fluorescence, the fluorescence intensity of an undamaged site in the same nuclei was subtracted from the fluorescence intensity of the accumulation spot for every cell at each time point. Nonspecific photobleaching and UV lamp output fluctuation were compensated for by correcting the accumulation site fluorescence intensity (IN) of each time point based on pre-laser background intensity using the formula: IN(t) = Idt/Ibt*IbpreIR, where Idt represents the difference between the accumulation spot intensity and the undamaged site background intensity of each time point, Ibt represents the background intensity of each time point, and IbpreIR represents the background intensity before irradiation.

U2OS cells were co-transfected with plasmids expressing GFP-ATRIP (either GFP-ATRIPWT or GFP-ATRIP2KR) and DsRed-PCNA (as a marker of DNA damage sites), and subjected to laser damage. Ten cells positive for PNCA recruitment were analyzed and the fluorescence intensity of GFP-ATRIP was measured up to 60 min. Each data point is the average of 10 independent measurements and error bars represent SEM (n=10).

Sequences of siRNAs

ATRIP 3’ UTR siRNA: / CACCACUCCUUUCCUUACCACAUCA
UBC9-1 siRNA: / ACUCCGUGGGAAGGAGGCUUGUUUA
UBC9-2 siRNA: / GCUCAAGCAGAGGCCUACACGAUUU
UBC9-3 siRNA: / AGCCAAGAAGUUUGCGCCCUCAUAA
Control siRNA / GGGUAUCGACGAUUACAAA[dT][dT]

Supplemental Figure Legends

Fig. S1. UBC9 is required for the efficient activation of the ATR pathway. (A) U2OS cells were transfected with control, UBC9-1, or UBC9-2 siRNA. At 50 hr after transfection, cells were labeled with EdU and analyzed for DNA synthesis. (B-C) HeLa cells were transfected with control, UBC9-1, or UBC9-2 siRNA. At 50 hr after transfection, cells were irradiated with UV or labeled with EdU. The effects of UBC9 knockdown on Chk1 phosphorylation were analyzed in (B), and the effects on DNA synthesis were analyzed in (C). (D) HeLa cells were transfected with siRNAs and treated with IR (5 Gy). The levels of the indicated proteins and phosphorylated Chk2 were analyzed by Western 1 hr after irradiation.

Fig. S2. Characterizations of ATRIP SUMOylation. (A) The sequences of the three putative SUMOylation sites of human ATRIP and the sequence conservation of these sites in higher vertebrates are shown. (B-C) HeLa cells transiently expressing SFB-tagged ATRIPWT, ATRIP2KR, or carrying the vector were subjected to immunoprecipitation using anti-Flag antibody under a denaturing condition. SUMOylated ATRIP was analyzed by Western blot using SUMO1 or SUMO2/3 antibody. (D) HeLa cells were treated with 1 mM HU for 5 hr or mock treated, and subjected to immunoprecipitation using control or SUMO2/3 antibody under a denaturing condition. SUMOylated ATRIP was detected using ATRIP antibody. (E) HeLa cells were transfected with plasmids expressing HA-SUMO2 and SFB-ATRIPWT or its indicated mutant derivatives. SFB-ATRIP proteins were immunoprecipitated using anti-Flag antibody under a denaturing condition. The levels of SUMOylated SFB-ATRIP and its mutant derivatives were analyzed using SUMO2/3 antibody. (F) A GST-tagged ATRIP fragment (amino acids 214-328) and its derivative containing the K234R and K289R mutations were expressed in E. coli and purified. Purified GST-ATRIP fragments were subjected to in vitro SUMOylation assays in the presence of E1, E2, SUMO2, and ATP. SUMOylated ATRIP fragments were detected by Western blot using SUMO2/3 antibody.

Fig. S3. ATRIP SUMOylation is critical for ATR activation. (A) HeLa cells stably expressing SFB-ATRIPWT and SFB-ATRIP2KR were treated with ATRIP 3’ UTR siRNA. Three days later, cells were labeled with EdU and analyzed for DNA synthesis. Error bars: S.D. (n=3). (B) HeLa cells stably expressing SFB-ATRIPWT, SFB-ATRIP2KR (clone 2KR-7), or carrying the vector were transfected with control or ATRIP 3’ UTR siRNA and irradiated with UV (8 J/m2). The levels of the indicated proteins and phosphorylated Chk1 were analyzed by Western 1 hr after UV treatment. (C) HeLa cells stably expressing SFB-ATRIPWT or SFB-ATRIP2KR were transfected with ATRIP 3’ UTR siRNA and irradiated with IR (10 Gy) 3 days later. One hour after irradiation, cells were labeled with EdU for 30 min, and the fractions of highly EdU-positive cells were quantified. Error bars: S.D. (n=2). (D) SFB-tagged ATRIPWT and ATRIP2KR were transiently expressed in HeLa cells and immunoprecipitated using anti-Flag antibody. The precipitated SFB-ATRIP was digested with increasing amounts of proteinase K (0, 3, 10, 30, and 100 ng) and analyzed by Western using anti-Flag antibody.

Fig. S4. Expression of GFP-ATRIPWT and GFP-ATRIP2KR in HeLa cells. GFP-ATRIPWT and GFP-ATRIP2KR were transiently expressed in HeLa cells at similar levels. The localizations of GFP-ATRIPWT and GFP-ATRIP2KR were analyzed in Fig. 4A-B.

Fig. S5. UBC9 knockdown does not affect the activation of the ATM pathway. (A) HeLa cells transfected with control or UBC9-2 siRNA were irradiated with 10 Gy of IR or mock treated, and subjected to immunoprecipitation with ATM, NBS1, or control antibodies 1 hr after irradiation. The ATM and NBS1 in the input and immunoprecipitates were analyzed using the respective antibodies. (B) HeLa cells extracts were incubated with purified GST-1xSUMO2 and GST-4xSUMO2. After GST-tagged SUMO2 was retrieved with glutathione beads, the presence of ATM, Chk2, MDC1, and 53BP1 in the pulldowns was tested using respective antibodies.

Fig. S6. Fusion of a SUMO2 chain to ATRIP2KR partially suppresses its defects in localization and ATR activation. (A) HeLa cells stably expressing SFB-ATRIP3S-2KR were treated ATRIP 3’ UTR siRNA and control or UBC9-2 siRNA. The localization of SFB-ATRIP3S-2KR was analyzed 30 min after laser microirradiation. The fractions of RPA32 stripe-positive cells that displayed ATRIP stripes were quantified. Error bars: S.D. (n=3). (B) SFB-ATRIPWT and SFB-ATRIP2KR were immunoprecipitated with anti-Flag antibody. The endogenous ATRIP coprecipitated with SFB-ATRIP was analyzed by Western. (C) HeLa cells stably expressing SFB-tagged ATRIPWT, ATRIP2KR, ATRIP3S-2KR, or carrying the vector were transfected with control siRNA and irradiated with UV (50 J/m2). The phosphorylation of Chk1 and RPA32 was analyzed 1 hr after UV treatment. Because the levels of total Chk1 were lower in the ATRIP2KR-expressing cell line than in other cell lines, the last 4 samples were normalized to Chk1 levels and re-analyzed.

References

Uematsu, N., Weterings, E., Yano, K., Morotomi-Yano, K., Jakob, B., Taucher-Scholz, G., Mari, P.O., van Gent, D.C., Chen, B.P., and Chen, D.J. (2007). Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks. J Cell Biol 177, 219-229.