Supplementary Information for. Regulation of Rev1 by the Fanconi Anemia Core Complex

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1 Supplementary Information for Regulation of Rev1 by the Fanconi Anemia Core Complex Hyungjin Kim, Kailin Yang, Donniphat Dejsuphong, Alan D. D Andrea* *Corresponding Author: Alan D. D Andrea, M.D. Alan_dandrea@dfci.harvard.edu This pdf file includes: Supplementary figure 1 to 6 Supplementary method References

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3 Supplementary Figure 1 Characterization of C1orf86 protein (Related to Fig. 1) (a) Protein sequence alignment of C1orf86 homologues from multiples species. C1orf86 is conserved in human, mouse, cow, and chicken. Sequence alignment was performed using ClustalW2. * (asterisk) indicates fully conserved residues, while a : (colon) and a. (period) indicate conservation between strongly similar and weakly similar amino acid groups, respectively. Amino acid residues were colored according to their physicochemical properties. (b) (Left) purification of GST-ubiquitin. GST and GST-ubiquitin were expressed in BL21 bacteria and purified using glutathione-sepharose beads. 10 % of input was visualized by coomassie staining. (Right) C1orf86 wild-type, but not the UBZ4 mutant, binds to ubiquitin. Purified GST or GST-Ub was incubated with in vitro translated Flag-tagged C1orf86 wild-type or UBZ4 mutant (C147A C150A) and immunobloted with anti-flag antibody. (c) Knockdown efficiency of C1orf86 sirna duplexes. HeLa cells were serially transfected with two independent sirnas against C1orf86 and pcdna3-flag-c1orf86, and cell lysates were analyzed with indicated antibodies. (d) C1orf86 depletion compromises FANCD2 monoubiquitination. HeLa cells treated with two independent sirnas against C1orf86 were left untreated or treated with 50 ng ml -1 MMC for 17 h, and cell lysates were analyzed by immunoblotting. (e) Representative pictures of metaphase spreads of sirna-transfected 293T cells exposed to 20 ng ml -1 MMC are shown. Arrows indicate examples of chromosome breaks and radial chromosomes in C1orf86-depleted cells. 3

4 Supplementary Figure 2 Stabilization of the FA core complex by FAAP20 (Related to Fig. 2) (a) Negative control for in vitro interaction. Myc-FAAP20 and FANCG were in vitro transcribed and translated, and the protein mixture was subjected to anti-myc immunoprecipitation and immunoblot analyses. (b) Verification of sirna that targets 3 UTR of FAAP20 mrna. HeLa cells transfected with sirna against FAAP20 were left untreated or treated with 50 ng ml -1 MMC for 17 h and the cell lysates were analyzed by indicated immunoblotting. (c) HeLa cells stably expressing Flag-HA-FAAP20 were transfected with indicated sirna and analyzed by indicated immunoblotting. Although sirna #3 impairs FANCD2 activation as shown in (b), it does not deplete exogenous protein encoded by FAAP20 cdna. 4

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6 Supplementary Figure 3 Characterization of the FAAP20: Rev1 interaction (Related to Fig. 3) (a) FAAP20 associates with Rev1. Myc-tagged FAAP20 was transiently coexpressed with Flagtagged Rev1, and cell lysates were incubated with anti-myc conjugated beads, and immunoprecipitated proteins were analyzed by immunoblotting. Anti-Flag immunoprecipitates from Flag-Rev1 expressing cells were run in parallel to identify the migrating position of Flag- Rev1. (b) Flag-FAAP20 and GFP-Rev1 were coexpressed in U2OS cells, and cell lysates were subjected to anti-flag immunoprecipitation. FANCA and FANCD2 were immunoblotted for positive and negative controls, respectively. (c) The UBZ4 domain of FAAP20 is required for the interaction with Rev1. Flag-tagged Rev1 was transiently coexpressed with GFP-tagged FAAP20 wild-type, or UBZ4 domain (C147A C150A; CA) mutant in 293T cells, and cell lysates were subjected to anti-flag immunoprecipitation and immunoblot analyses. (d) Rev1 is monoubiquitinated in vivo. GFP-tagged Rev1 was expressed alone or coexpressed with HAubiquitin in 293T cells, and cell lysates were analyzed by anti-gfp and anti-rev1 immunoblots. Slower-migrating GFP-Rev1 band represents the monoubiquitinated isoform. (e) Rev1 undergoes deubiquitination. 293T cell lysates coexpressing Flag-Rev1 and HA-ubiquitin were subjected to anti-flag immunoprecipitation. In parallel, cells expressing Flag-Rev1 were lysed in ubiquitin-aldehyde (Ub-aldehyde) to inhibit deubiquitination before anti-flag immunoprecipitation. Slower-migrating form of Flag-Rev1 indicates the conjugation of endogenous ubiquitin to Flag-Rev1. 6

7 Supplementary Figure 4 Verification of the interaction between Rev1-monoubiquitin and FAAP20 (Related to Fig. 4) (a and b) The blots used in Fig. 4b and Fig. 4c were stripped and reprobed with anti-ha antibody to detect the Rev1-conjugated HA-tagged ubiquitin coimmunoprecipitated with myc- FAAP20. 7

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9 Supplementary Figure 5 Characterization of FAAP20 foci (Related to Fig. 5) (a) GFP-FAAP20 forms foci in the absence of FANCA and FANCD2 ubiquitination. Patientderived FANCA-deficient (FANCA ) or cdna corrected (FANCA+) GM6914 fibroblasts were transfected with GFP-FAAP20 on the coverslip, and GFP-fluorescence was examined after fixation. (b) PCNA is monoubiquitinated in response to crosslinking agent MMC. U2OS cells were treated with 40 J m -2 UVC or 1 µg ml -1 MMC for indicated times and the cell lysates were analyzed by indicated immunoblotting. (c) FAAP20 and Rev1 colocalize with PCNA at the sites of MMC-induced DNA lesion. U2OS cells transfected with GFP-tagged Rev1 or FAAP20 was treated with 1 µg ml -1 MMC for 18 h and stained with anti-pcna antibody. 9

10 Supplementary Figure 6 Mutation spectra of UVC-irradiated supf gene (Related to Fig. 5) (a) White colonies that harbor damaged supf plasmids derived from sirna control or FAAP20 cells were re-streaked, and the isolated plasmids were subjected to DNA sequencing. The percentage of nucleotide transitions and transversions in the structural supf gene is shown. 10

11 Supplementary method Description of the bioinformatic search to identify novel UBZ4-containing proteins UBZ4 domain-containing proteins were identified by the Hidden Markov Model (HMM) using C2HC Zn-finger sequences signature as bait as previously described 1. For this analysis, we used the conserved sequences of UBZ4 domain of Polκ from multiple species 2, and first built a Hidden Markov Model (HMM) of a consensus sequence using the algorithm HMMER 3. We generated a protein expression database containing all known protein sequences from the following species, Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, and Arabidopsis thaliana, downloaded from ExPASy database 4. All protein sequences were then reversed to generate a control database. The algorithm HMMER was used to perform Hidden Markov search with the UBZ4 consensus model, in both protein expression database and control database. Hits with significant statistical value only in the protein expression database but not in the control database were selected further detailed sequence analysis. Among the most interesting hits, we found RAD18, SLX4, FAN1, and SNM1A, recently identified UBZ4-containint proteins involved in ICL repair and translesion synthesis. We further identified ten uncharacterized human proteins. Based on the availability of sirnas, we knocked down five of these in HeLa cells. sirnas for two of these genes caused a knockdown of FANCD2-monoubiquitination levels. But, only knockdown of one gene (C1orf86; Q6NZ36_Human) caused a reduction in MMC-inducible FANCD2-Ub. Therefore, we focused on the C1orf86 gene in our paper. References 1. Yang, K., Moldovan, G.L. & D'Andrea, A.D. RAD18-dependent recruitment of SNM1A to DNA repair complexes by a ubiquitin-binding zinc finger. J Biol Chem 285, (2010). 2. Bienko, M. et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, (2005). 3. Eddy, S.R. Profile hidden Markov models. Bioinformatics 14, (1998). 4. Gasteiger, E. et al. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31, (2003). 11