Fig 2: Model for the Aurora B-RPA signaling axis.During mitosis, RPA bound to ssDNA intermediates or free in solution is phosphorylated by Aurora B kinase at Ser-384 (DBD-B) in the large RPA70 subunit. We propose that phosphorylation releases the protein-interaction domains (OB-F or PID70N and wh or PID32C) and promotes the formation of higher-density RPA-bound ssDNA filaments. The protein-interaction domains can then promote binding to RPA-interacting proteins. The feedback mechanism that modulates Aurora B activity through RPA phosphorylation and the involvement of R loops also remains to be elucidated. Since the site of phosphorylation resides close to the DSS1 binding site, recruitment of DSS1-BRCA2 is inhibited leading to suppression of homologous recombination during mitosis. Deregulation of the Aurora B-RPA signaling circuit leads to errors in chromosome segregation fidelity.

Fig 3: Accumulation of lagging strand gaps in BRCA-deficient cells.a, b BrdU alkaline comet assay showing that PRIMPOL depletion reduces, but does not abolish replication stress-induced ssDNA accumulation in BRCA2-knockout HeLa cells, indicating the presence of PRIMPOL-independent gaps. Representative micrographs (a) and quantifications (b) are shown. At least 45 nuclei were quantified for each condition. The mean values are represented on the graph, and the p-values (t-test, two-tailed, unpaired) are listed at the top. A schematic representation of the assay conditions is also presented. RT-qPCR experiments confirming PRIMPOL depletion are presented in Supplementary Fig. 6a. c, d SIRF assay showing that, upon replication stress, LIG1 knockdown induces PAR chain formation in wild-type HeLa cells, but not in BRCA1 or BRCA2-depleted HeLa cells upon treatment with a low dose of HU (0.4 mM), indicating the prevalence of lagging strand gaps in these cells. Representative micrographs (scale bar represents 10 μm) (c) and quantifications (d) are shown. At least 35 positive cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. Western blots confirming the co-depletion are shown in Supplementary Fig. 6d. e, f SIRF assay showing that Polα inhibition suppresses the increased CHAF1A retention in BRCA1 or BRCA2-depleted HeLa cells. Representative micrographs (scale bar represents 10 μm) (e) and quantifications (f) are shown. At least 35 positive cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. g, h SIRF assay showing that RPA1 co-depletion restores HIRA levels to the same levels in wild-type and BRCA1 or BRCA2-knockodown HeLa cells. Representative micrographs (scale bar represents 10 μm) (g) and quantifications (h) are shown. At least 30 positive cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. Western blots confirming the co-depletion are shown in Supplementary Fig. 6h.

Fig 4: ADAR1 controls the recruitment of TOPBP1 to replication stress sites. (A) Immunostaining and confocal microscopy analysis of TOPBP1 and RPA1 foci formation in ADAR1-depleted U2OS cells. Cells were treated with CPT for 1 h followed by pre-extraction and fixation. The intensity of TOPBP1 foci was quantified and normalized to that of RPA1 foci (n > 200). Scale bars, 10 μm. (B) Immunostaining and confocal microscopy analysis of the recruitment of FLAG- ATRIP at replication stress sites in HeLa cells expressing siRNAs against ADAR1. Cells were treated with CPT for 1 h followed by pre-extraction and fixation. The intensity of ATRIP foci was quantified and normalized to that in control cells (n > 100). Scale bars, 10 μm. (C, D) Immunostaining and confocal microscopy analysis of TOPBP1 and RPA1 foci formation in HeLa cells expressing ADAR1 5′UTR siRNA and the indicated stably integrated ADAR1 variants. Cells were treated with CPT for 1 h before collection. The intensity of TOPBP1 foci was quantified and normalized to that of RPA1 foci (n > 200 for C; n > 110 for D). Scale bars, 10 μm. (E) Co-IP analysis of the interaction between TOPBP1 and RAD9 in control or ADAR1-knockdown HeLa cells. (F) Co-IP analysis of the interaction between TOPBP1 and RAD9 in HeLa cells expressing ADAR1 5′UTR siRNA and the indicated ADAR1 variants. (G) Pull-down assays with biotinylated RAD9 pS387 peptides and the indicated recombinant proteins. ADAR1 RBDs/Wt or RBDs/EAXXA was incubated with TOPBP1 BRCT 0–2 followed by addition of the biotinylated RAD9 pS387 peptides. The protein complexes were then captured with streptavidin beads for immunoblotting. Data are mean ± SDs for (A–D) from biological triplicate experiments. **P < 0.01; NS, not significant; Kruskal–Wallis test for (A–D).

Fig 5: Defects in chromosome segregation induced by loss of Ser-384 RPA70 phosphorylation.Representative immunofluorescent images stained with DAPI and anti-Tubulin antibody depict anaphase bridges (white arrow) in the mitotic population of asynchronous RPASA/SA mutant and RPAWT/WT cells (a) and in cells released from prometaphase arrest (b). Images are representative of three independent experiments. Quantitation of anaphase bridges and lagging chromosomes in mitotic population of asynchronous cells (c) and in cells released from prometaphase arrest (d) RPAWT/WT and RPASA/SA cells. More than 70 cells were counted from three independent experiments. Data represent SEM and statistical significance was determined using an unpaired two-tailed t-test. ***p = 0.0001 (c) and *** p = 0.0002 (d).