Fig 2: Preventing FA pathway activation alleviates ICL hypersensitivity of SCAI‐deficient cells Schematic outline of genome‐scale CRISPR‐Cas9 screen for genes whose KO suppresses MMC hypersensitivity of SCAI KO cells. LD80, 80% lethal dose; NGS, next‐generation sequencing.MaGECK analysis of sgRNA enrichment in CRISPR screen in (A) following MMC treatment (n = 3 technical replicates; false discovery rate (FDR) of 0.2 indicated by dotted line). FA genes are highlighted in blue.GO term analysis of significantly enriched genes (FDR < 0.2) in CRISPR‐Cas9 screen in (A), using Reactome pathways from PANTHER16.0.Immunoblot analysis of FANCA siRNA knockdown efficiency in U2OS cells.Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non‐targeting control (CTRL) or FANCA siRNAs and subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later, and co‐stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; *P < 0.05; ns, not significant, two‐tailed paired t‐test).As in (F), except that cells were stained with γH2AX antibody (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; **P < 0.01; ns, not significant, two‐tailed paired t‐test).Immunoblot analysis of the indicated U2OS cell lines.Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI double KO (DKO) cells subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.

Fig 3: The proteasome inhibitor Bortezomib inhibits not only FANC signaling, but also HR, Alt-EJ, and end resection.(A) Bortezomib treatment disrupts FANCD2 IRIF. U2OS cells were treated with Bortezomib (100 nM, 4 hr before IR treatment) or vehicle (DMSO) as well as with an individual FANCA siRNA (siFANCA-3) or non-targeting siRNA (siCTRL). Subsequently, cells were treated with 10 Gy IR and allowed to recover (4 hr) prior to fixation and immunostaining for FANCD2 and the DSB marker γH2AX. Cells positive for γH2AX foci were scored for FANCD2 foci. Shown is the percentage of U2OS cells with >20 FANCD2 foci for each treatment. *P≤0.01 (n≥3). (B) Bortezomib treatment causes a greater reduction in Alt-EJ, HDR, and SSA, relative to Distal-EJ. The U2OS reporter cell lines were treated with siCTRL prior to transfection with expression vectors for I-SceI, or a GFP expression vector in parallel to quantify percent transfected cells, and treated with Bortezomib (17 nM) or vehicle (DMSO). The concentration of Bortezomib in these experiments was lower than that used in A, since the reporter assays require a dose that is sublethal for several days of continual treatment. Shown are %GFP+ frequencies normalized to transfected cells, and compared to vehicle (DMSO) treated. *P≤0.004 distinct from the effect on Distal-EJ (n≥5). (C) Bortezomib treatment inhibits Alt-EJ independently of FANCA, FANCD2 and DNA2. The EJ2-GFP reporter assay was performed as in B for cells pre-treated with siRNAs targeting FANCA, DNA2 and FANCD2. Shown are the GFP+ frequencies normalized as in B. *P≤0.009 distinct from vehicle (DMSO) treated (n≥6). (D) Bortezomib treatment causes a marked decrease in end resection but does not affect cell cycle phase distribution. U2OS cells were treated with Bortezomib as in A prior to end resection analysis as in Fig. 6E. Shown are representative flow cytometry plots for the end resection assay, and the percentage of cells with chromatin-bound RPA staining quantified using this assay. *P<0.0001 (n≥3). Also shown are cell cycle phase percentages following the same Bortezomib treatment as for the end resection assay, based on BrdU labeling and PI staining as in Fig. 2C.

Fig 4: Protein phosphatase 2A dephosphorylates FAAP20 at the CPD motif.(A) U2OS cells expressing Flag-FAAP20 were left untreated or treated with 100 nM okadaic acid (OA) for 2 h, and FAAP20 pS113 levels were analyzed by WB using anti-pS113 antibody. (B) (Left) U2OS cells were serially transfected with siRNA control or PP2Ac and Flag-FAAP20-encoding plasmid, and FAAP20 pS113 levels were analyzed by WB. (Right) confirmation of PP2Ac knockdown by RT-qPCR. mRNA expression was normalized by GAPDH mRNA (mean ± SD; n = 3 independent experiments). * P <0.001, Student’s t-test. (C) U2OS cells were transfected with indicated plasmids, and FAAP20 pS113 levels were analyzed by WB. (D) 293T cells transfected with HA-B56α and Flag-FAAP20 WT or N-terminal deletion mutants (ΔN30: a.a.1-30 deletion, ΔN48: a.a.1-48 deletion, and ΔN63: a.a.1-63 deletion) were subjected to anti-Flag IP and WB. * IgG heavy chain. (E) Summary of the binding regions for B56α, FANCA, and PIN1 revealed in this study.

Fig 5: Interactions between FANCA CTD mutants and FANCG CTD and their mutational effects in nuclear localization of FANCA (A) Yeast two hybrid analyses between deletion mutants of FANCA CTD and FANCG CTD. Master plate (SD-LeuTrp) and a selective plate (SD-LeuTrpHis containing 10 mM of 3-AT) are shown on the left and right, respectively. (B) Yeast two hybrid analyses between FANCA CTD with selected point mutants and FANCG CTD. Master plate (SD-LeuTrp) and a selective plate (SD-LeuTrpHis containing 10 mM of 3-AT) are shown on the left and right, respectively. Structures of the point mutants are shown in Figure 4. (C) FANCA-knockout U2OS cells expressing indicated mutants in the presence of 1uM MMC were immunostained to visualize FANCA. Cell nuclei were detected with 4′,6-diamidino-2-phenylindole (DAPI).