Legends for Supplemental Figures.

Transcription

1 1 Legends for Supplemental Figures. Supplemental Figure 1. Proteasome Inhibition does not abrogate the expression of FA core complex proteins or formation of the FA core complex. (Full-length blots are presented in Supplemental Fig. 3L-N) (A) Simplified model of the FA pathway. Eight FA proteins (FANCA, B, C, E, F, G, L and M), FAAP24 and FAAP100 form a multi-subunit ubiquitin ligase complex (FA core complex). In response to DNA damage and during normal S phase progression, the FANCD2 protein is monoubiquitinated on lysine 561 in an FA core complex (E3 ubiquitin ligase)- and UBE2T (E2 ubiquitin conjugating enzyme)-dependent manner. ATR kinase is required for increase of FANCD2 monoubiquitination in response to DNA damage. Monoubiquitinated FANCD2 is targeted to nuclear foci where it colocalizes with γh2ax, ATR, RPA, the MRN complex, BRCA1, BRCA2, RAD51, PALB2, FANCJ and other proteins. BRCA1 is required for nuclear foci formation of FANCD2 in response to DNA damage. All of these factors are required for cellular resistance to DNA crosslinking agents. On the other hand, PCNA is monoubiquitinated on K164 by RAD6 and RAD18, and this monoubiquitination activates translesion synthesis. The deubiquitinating enzyme USP1 negatively regulates monoubiquitination of both FANCD2 and PCNA. (B) Treatment with proteasome inhibitors did not affect the expression of FA core complex proteins. HeLa cells were exposed to IR (15Gy) and incubated in the presence of MG132 (2µM) for the indicated time. Expression of FANCA, C, D2, F, and G was examined by western blotting. Asterisks (*) indicate non-specific bands.

2 2 (C) Expression of FA core complex proteins and UBE2T was not affected by treatment with proteasome inhibitors. HeLa cells were exposed to IR (15Gy) and incubated in the presence of MG132 (2µM) for 8 hours. Expression of FANCA, C, D2, E, F, G, M and UBE2T was examined by western blotting. (D) Treatment with proteasome inhibitors did not affect the formation of FA core complex. HeLa cells were exposed to IR (15Gy) and/or incubated in the presence of MG132 (2µM) for 8 hours. A FANCA antibody was used to isolate immunocomplexes from whole cell extracts. IP products were analyzed by western blotting using FANCA, C and G antibodies. FANCC and FANCG co-immunoprecipitated with FANCA in all conditions. Supplemental Figure 2. Analysis of ATR and ATM activation after DNA damage in the presence of proteasome inhibitors. (Full-length blots are presented in Supplemental Fig. 3O-Q) (A) Proteasome inhibition did not inhibit cisplatin-, HU- and UV-induced CHK1 phosphorylation, while it inhibited RPA2 phosphorylation and FANCD2 monoubiquitination. HeLa cells were incubated in cisplatin (CDDP 25µM) or hydroxyurea (1mM) for 24 hours, or exposed to UV radiations (50J/m2), and incubated in the presence of bortezomib for 8 hours. FANCD2, CHK1, CHK1-Ser345P and RPA2 immunoblots were performed using whole cell extracts. (B) Proteasome inhibition inhibits IR-induced CHK1 and RPA2 phosphorylations. HeLa cells were incubated in the presence of bortezomib (1µM) for 4 hours, irradiated (15Gy)

3 3 and incubated for the indicated time after irradiation. FANCD2, CHK1, CHK1-Ser345P and RPA2 immunoblots were performed using whole cell extracts. (C) Proteasome inhibition does not inhibit IR-induced phosphorylation of ATM substrates. HeLa cells were treated as described in (B). ATM-dependent phosphorylations of ATM Ser1981, NBS1 Ser343, CHK2 Thr68, and FANCD2 Thr691 in response to IR were analyzed by western blotting. Supplemental Figure 3. Non-cropped pictures of blots and gels. The Western-blot membranes were cut to allow multiple simultaneous hybridizations. (A-D) Non-cropped pictures of blots presented in Figure 1. (E and F) Non-cropped pictures of gels and blots presented in Figure 2. (G-J) Non-cropped pictures blots presented in Figure 3. (K) Non-cropped pictures of blot presented in Figure 6. (L-N) Non-cropped pictures of blot presented in Supplemental Figure 1. (O-Q) Non-cropped pictures of blot presented in Supplemental Figure 2. Material and Methods for Supplemental Figures Western blot analysis. Mouse monoclonal antibodies directed against ATM- Ser1981P ( , Rockland, 1/2000), CHK1 (G-4, Santa Cruz, 1/1000), RPA2 (Ab-2, Calbiochem, 1/1000), rabbit polyclonal antibodies against ATM (GTX17955, Genetex, Inc., 1/5000), phospho-chk1 (Ser345) (#2341, Cell Signaling, 1/500), CHK2 (07-057, Upstate, 1/1000), phospho-chk2 (Thr68) (#2661, Cell Signaling, 1/1000), FANCA (#7488, a gift of Dr. Maureen Hoatlin, 1/1000), FANCC ((1), 1/500), phosho-

4 4 FANCD2 (Thr691) ((2), 1/500), FANCE (gift of Drs. XiaoZhe Wang and Alan D Andrea, 1/2000), FANCF ((3), 1/1000), FANCG ((4), 1/1000), FANCM ((5), gift of Dr. Weidong Wang (National Institute on Aging), 1/2000), and UBE2T (BC004152, ProteinTech Group (Chicago, IL), 1/2000) were used as a primary antibodies. Immunoprecipitation. Using a polyclonal antibody to FANCA (#7488, gift from Dr. Maureen Hoatlin, Oregon Health and Science University), immunoprecipitation was performed as previously described (6). References for supplemental data 1. Yamashita T, Barber DL, Zhu Y, Wu N, D'Andrea AD. The Fanconi anemia polypeptide FACC is localized to the cytoplasm. Proc Natl Acad Sci U S A 1994; 91: Ho GP, Margossian S, Taniguchi T, D'Andrea AD. Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance. Mol Cell Biol 2006; 26: Siddique MA, Nakanishi K, Taniguchi T, Grompe M, D'Andrea AD. Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells. Exp Hematol 2001; 29: Garcia-Higuera I, Kuang Y, Naf D, Wasik J, D'Andrea AD. Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. Mol Cell Biol 1999; 19:

5 5 5. Meetei AR, Medhurst AL, Ling C, et al. A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nat Genet 2005; 37: Taniguchi T, D'Andrea AD. The Fanconi Anemia Protein, FANCE, Promotes the Nuclear Accumulation of FANCC. Blood 2002; 100:

6

7

8