Fig 2: SCAI prevents Polθ‐mediated MMEJ during ICL repair (related to Fig 5) Whole cell extracts of U2OS and U2OS/SCAI KO cells were analyzed by immunoblotting with indicated antibodies.Mock‐, 53BP1‐, or SCAI‐C‐depleted egg extracts were analyzed by immunoblotting with indicated antibodies.pICLpt was replicated in extracts immunodepleted in mock‐, 53BP1‐, or SCAI‐C‐depleted egg extracts digested with PsiI+XhoI and resolved on a denaturing polyacrylamide gel.pICLpt was replicated in SCAI‐C‐depleted extracts that were also depleted of BRCA2, or supplemented with DNA‐PKcs inhibitor (NU7441; 100 µM) where indicated. Reaction samples were digested with PsiI+XhoI and analyzed on a denaturing polyacrylamide gel.Mock‐ or BRCA2‐depleted egg extracts were analyzed by immunoblotting with indicated antibodies.Most frequent deletion products in sequencing data in Fig 5A. In red, crosslinked guanines; highlighted in grey, deletions; underlines, microhomology regions.Schematic representation of DNA synthesis occurring across the adducted base following DNA incisions on either the top or bottom strand. The denoted nucleotide position 1,548 corresponds to the insertion 0 position when incisions occur in the bottom strand or to the −1 position when incisions occur in the top strand. Sequencing products of correct length from Fig 5A were analyzed for their nucleotide misincorporation distribution (bottom graphs). Misincorporation is based on the bottom strand read.Samples in Fig 5C were analyzed by native agarose gel electrophoresis.pICLpt was replicated in mock‐ or SCAI‐depleted extracts treated or not with Novobiocin (NVB; 150 µM) in the presence of [α‐32P]dATP, and reactions were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled.Samples in (I) were digested with PsiI+XhoI or AflIII and analyzed on a denaturing polyacrylamide gel.Clonogenic survival of U2OS and U2OS/SCAI KO cells treated or not with Novobiocin (NVB; 50 µM) and subjected to various doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).

Fig 3: Model of SCAI function in ICL repair via the FA pathwaySee main text for details.

Fig 4: SCAI‐deficient cells display major hallmarks of FA gene dysfunction (related to Figs 1 and 2) GO term analysis of significant hits (NormZ < −3) in CRISPR‐Cas9 screen in Fig 1A, using Reactome pathways from PANTHER16.0.Clonogenic survival of U2OS WT and U2OS/SCAI KO cells subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).As in (B), except that cells were treated with indicated doses of cisplatin for 24 h (mean ± SEM; n = 3 independent experiments).Scatter plot showing cell cycle distribution of cells in Fig 1F. Light grey, G1 phase; dark grey, S phase; red: G2/M phase. Proportion of cells in G2/M phase is indicated.U2OS WT, U2OS/SCAI KO, and U2OS/SCAI KO/Strep‐HA‐SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later, and co‐stained with 53BP1 antibody and DAPI. 53BP1 foci were quantified by QIBC (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; *P < 0.05; two‐tailed paired t‐test).As in (E), except that cells were stained with RAD51 antibody (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; *P < 0.05; ns, not significant, two‐tailed paired t‐test).Representative images of the experiments in Fig 1G and H. Scale bar, 10 µm.Representative images of the experiments in (E) and (F). Scale bar, 10 µm.Immunoblot analysis of FANCD2 siRNA knockdown efficiency in U2OS cells.Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non‐targeting control (CTRL) or FANCD2 siRNAs and subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).Immunoblot analysis of U2OS WT and U2OS/SCAI KO cells harvested at the indicated times after exposure to MMC (0.5 µM).

Fig 5: SCAI interacts with Polζ but protects against erroneous ICL repair independently of Polζ Analysis of SCAI‐interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI‐C and SCAI‐N immunoprecipitates (IPs) compared to an IgG control, plotted against the P‐value (n = 4 independent experiments; permutation‐based FDR < 0.01, s0 = 2).SCAI, REV7, REV1, and REV3 immunoprecipitates (IP) in NPE were immunoblotted with the indicated antibodies. Asterisks denote antibody cross‐reactivity.Whole proteome MS analysis of mock‐ versus SCAI‐depleted egg extracts. The volcano plot shows the difference in abundance of proteins between the mock reaction and SCAI‐depleted samples (with either N or C antibodies) (X‐axis), plotted against the P‐value resulting from two‐tailed Student’s t‐test (Y‐axis). Proteins significantly down‐regulated (FDR < 5%) in SCAI‐depleted extracts are highlighted in red (n = 4 biochemical replicates; FDR < 5% corresponds to a permutation‐based FDR‐adjusted q‐value of < 0.05).GFP IPs from U2OS or U2OS/GFP‐SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP‐SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1‐Polζ complex that are independent of REV3.Schematic of SCAI interaction with the REV1‐Polζ complex. The SCAI‐interacting region of human REV3 and its alignment with a homologous region in human AHDC is indicated. NTD, N‐terminal domain; PCD, positively charged domain; Pol, polymerase domain; CTD, C‐terminal domain.GFP IPs from U2OS cells transfected with indicated REV3‐GFP expression constructs were immunoblotted with indicated antibodies.As in (F), except that U2OS or U2OS/GFP‐SCAI cells were transfected with FLAG‐NLS‐REV3(995–1194) expression plasmid.Mock‐, SCAI‐, REV1‐, or SCAI/REV1‐depleted egg extracts were analyzed by immunoblotting with indicated antibodies. Asterisks denote antibody cross‐reactivity.pICLpt was replicated in egg extracts immunodepleted with the indicated antibodies and processed as in Fig 3J and L.pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pull down, and associated proteins were analyzed by immunoblotting.