Fig 2: CHAF1A depletion is epistatic with PrimPol depletion and results in a decrease in PrimPol localization to replication forks. (A and B) BrdU alkaline comet assay showing that loss of PrimPol is epistatic with loss of either CHAF1A or ASF1A in (A) BRCA2KO or (B) WT HeLa cells. Accumulation of ssDNA gaps in samples depleted of either CHAF1A or ASF1A show no further decrease in ssDNA gaps when PrimPol is also depleted. At least 49 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown. (C) S1 Nuclease DNA fiber combing assay showing that CHAF1A co-depletion with PrimPol in WT or BRCA2KO HeLa cells does not result in a further decrease in ssDNA gap accumulation upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. (D) BrdU alkaline comet assay showing that loss of PrimPol is epistatic with loss of either CHAF1A or ASF1A in MDA-MB-436 cells. Accumulation of ssDNA gaps in samples depleted of either CHAF1A or ASF1A show no further decrease in ssDNA gaps when PrimPol is also depleted. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is displayed. (E) S1 Nuclease DNA fiber combing assay showing that CHAF1A or ASF1A co-depletion with PrimPol in MDA-MB-436 cells does not result in a further decrease in ssDNA gap accumulation upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. (F and G) BrdU alkaline comets showing the overexpression of PrimPol in BRCA2KO HeLa cells under 0.4 mM HU (F) and 150 μM cisplatin (G) treatment is able to partially rescue ssDNA gap abundance when CHAF1A is knocked down. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are displayed. (H) S1 Nuclease DNA fiber combing assay showing that overexpression of PrimPol in BRCA2KO HeLa cells is not able to rescue ssDNA gap accumulation to an observable level upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. (I and J) SIRF experiment showing that the increase in PrimPol localization to nascent DNA upon 0.4 mM HU treatment in BRCA2KO HeLa cells is lost when either CHAF1A or ASF1A are depleted (I). Representative micrographs with scale bars representing 10 μm are shown (J). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). A schematic representation of the assay conditions is shown.

Fig 3: Suppression of ssDNA gaps upon CHAF1A loss is independent of H3 deposition at actively replicating DNA. (A) SIRF assay for MRE11 localization showing that BRCA2KO induced localization of MRE11 to nascent DNA under 0.4 mM HU treatment is abrogated upon depletion of CHAF1A or ASF1A in BRCA2KO HeLa cells. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). A schematic representation of the assay conditions is shown. (B) SIRF assay showing that increased MRE11 recruitment to nascent DNA in response to knockdown of BRCA1 and BRCA2 in WT 293T cells is no longer present when CHAF1A is lost in CHAF1AKO 293T cells under 0.4 mM HU treatment. At least 186 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). A schematic representation of the assay conditions is shown. (C) BrdU alkaline comet assay showing that inhibition of MRE11 via 150 μM mirin treatment is epistatic with the decrease in ssDNA gap accumulation observed with knockdown of CHAF1A or ASF1A in BRCA1KO RPE1 cells. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Cells were incubated with or without mirin during the BrdU incubation period as well as with their respective mirin conditions during the following 1 h chase as shown in the schematic representation of the assay conditions displayed. (D and E) SIRF experiment showing differential impact of CHAF1A and ASF1A depletions on H3 localization to nascent DNA. Knockdown of CHAF1A in BRCA2KO HeLa cells leads to a rescue of H3 localization to nascent DNA under 0.4 mM HU treatment, whereas knockdown of ASF1A under these same conditions result in no change from control (D). Representative micrographs with scale bars representing 10 μm are shown (E). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). A schematic representation of the assay conditions is shown. (F and G) SIRF assay showing that CHAF1A localization to nascent DNA is decreased after treatment with 0.4 mM HU upon ASF1A knockdown in both WT and BRCA2KO HeLa cells similarly to those with CHAF1A knocked down (F). Representative micrographs with scale bars representing 10 μm are shown (G). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). A schematic representation of the assay conditions is shown.

Fig 4: Loss of ASF1A results in chemoresistance in BRCA-deficient cells. (A) Neutral comet assay showing that DSBs in BRCA2KO HeLa cells induced by 150 μM cisplatin treatment for 2 h are suppressed with knockdown of ASF1A similarly to knockdown of CHAF1A. At least 47 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are displayed. (B) γH2AX immunofluorescence showing that γH2AX foci induced by 150 μM cisplatin treatment for 2 h in BRCA2KO HeLa cells are suppressed by knockdown of ASF1A similarly to knockdown of CHAF1A. At least 100 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unpaired). (C) Clonogenic survival assay showing that loss of ASF1A promotes cisplatin resistance in BRCA2KO HeLa cells. The average of three experiments, with SEM indicated as error bars, is shown. Asterisks indicate statistical significance (two-way ANOVA). (D) Table summarizing the observed consequences of CHAF1A or ASF1A loss in BRCA-deficient cells on FP, RGS, DSB accumulation and drug response status. (E) Schematic model of the proposed model of CAF-1 role in efficient PrimPol recruitment to the replication fork and ssDNA gap generation. Created in BioRender. Moldovan, G. (2024) BioRender.com/y25 × 850.

Fig 5: Co-depletion of EXO1 and CHAF1A reduces cellular viability. (A, C, E). Volcano plots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are presented based on their impact on the viability of EXO1KO#1 compared to WT cells (A), EXO1KO#3 compared to WT cells (C), and as control, EXO1KO#1 compared to EXO1KO#3 cells (E). Genes are plotted by the −log10 of their respective negative and positive P-values and associated log2 Fold Change values. The hit chosen for validation, namely CHAF1A, is indicated. (B, D, F) Scatterplots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are plotted based on their impact on the viability of EXO1KO#1 compared to WT cells (B), EXO1KO#3 compared to WT cells (D), and as control, EXO1KO#1 compared to EXO1KO#3 cells (F). The hit chosen for validation, namely CHAF1A, is indicated. (G) Table showing the ranks in the synthetic lethality screens, and the biological roles of CHAF1A. (H, I) Clonogenic survival assays showing that siRNA (H) and sgRNA (I) depletion of CHAF1A reduces the viability of EXO1-knockout cells compared to WT HeLa cells. Clonogenic survival is presented normalized to WT control cells. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t-test unpaired).