Fig 2: SCAI prevents Polθ‐mediated MMEJ during ICL repair pICLpt was replicated in egg extracts immunodepleted with indicated antibodies. Replication samples were amplified by PCR and analyzed by next‐generation sequencing for deletions and insertions. Results from two independent experiments are shown.Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross‐reactivity.pICLpt was replicated in extracts immunodepleted as in (B), digested with PsiI+XhoI or AflIII and resolved on a denaturing polyacrylamide gel.Immunoblot analysis of Polθ siRNA knockdown efficiency in U2OS cells.Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Fig 1I. Scale bars, 10 µm.Quantification of radial chromosomes in (E) (mean ± SD; 1,197, 241, 155, and 90 metaphase cells analyzed per condition (left to right) pooled from three independent experiments; *P < 0.05, two‐tailed t‐test).Quantification of chromosomal breaks/gaps in (E) (mean ± SEM; 150, 150, 114, and 90 metaphase cells analyzed per condition (left to right) pooled from three independent experiments; ****P < 0.0001, Mann–Whitney U test).Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non‐targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).

Fig 3: SCAI interacts with Polζ (related to Fig 4) Heat map depicting the mean of the Z‐scored Log2 label‐free quantification LFQ intensity from four biochemical replicates of pCTRL and pDPCLeads replication in Xenopus egg extracts (data originally published in Larsen et al (2019)). Where indicated, Geminin was added to block DNA replication, and ubiquitin‐vinyl sulfone (Ub‐VS) was added to deplete the pool of free ubiquitin from the extracts.pDPCLeads was replicated in mock‐, SCAI‐C‐, and SCAI‐N‐depleted egg extracts in the presence of [α‐32P]dATP. Reactions were analyzed by native agarose gel electrophoresis. Red arrowheads indicate accumulation of open circular (OC) molecules caused by a TLS defect.Nascent leading strand and extension products generated upon FspI+AatII digest of pDPC. Double digestion generates shorter damaged and longer undamaged extension products. The CMG helicase is depicted in green and the crosslinked M.HpaII protein in grey.pDPC was replicated in mock‐ or SCAI‐depleted Xenopus egg extract in the presence of [α‐32P]dATP. Samples were digested with FspI+AatII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the DPC site are indicated. U, undamaged; D, damaged extension product.U2OS cells or derivative lines stably expressing GFP or GFP‐SCAI were subjected to GFP IP followed by immunoblotting with indicated antibodies.U2OS WT or SCAI KO cells that were transfected with REV3‐GFP where indicated were subjected to IP with IgG or SCAI antibody followed by immunoblotting.GFP IPs from U2OS cells transfected with indicated GFP‐AHDC1 expression constructs were analyzed by immunoblotting.Alignment of the SCAI‐interacting regions of human and Xenopus REV3 proteins. Asterisk indicates positions with a conserved amino acid; colon indicates conservation between amino acids with strongly similar properties; period indicates conservation between amino acids with weakly similar properties.Recombinant GST or GST‐xSCAI proteins incubated or not with Biotin‐xREV3 peptide (aa 1110–1179) were subjected to streptavidin pull down and analyzed by Coomassie staining.

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 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).