Fig 2: Activation of the DNA damage response during lytic replication of KSHV in EA.hy926 cells and following RTA expression alone. Levels of phosphorylated H2AX and RPA32 following addition of doxycyline to control EA.hy926 cells, EA.hy926 cells containing an ORF50 expression construct (EA.hy926-RTA) and KSHV-infected EA.hy926 cells containing an ORF50 expression construct (rKSHV-EA.hy926-RTA). Expression of LANA and ORF57 confirm presence of KSHV and lytic reactivation of the virus respectively.

Fig 3: XAB2 depletion leads to the increased engagement of NHEJ in S/G2. (A) Quantification of the CRISPR–Cas9 mClover-LMNA1-HDR assay in control and XAB2-depleted U2OS cells. Data is representative of three independent biological repeats. Bars represent mean percentages of mClover-positive cells over the iRFP-positive population ± s.e.m. Significant differences between specified comparisons were assessed by unpaired t-test and are highlighted by stars (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001). Inset, efficiency of XAB2 depletion achieved by siRNA (siXAB2) as compared to a non-silencing control siRNA (siCTRL). a-Tubulin was used as a loading control. (B and C) Representative immunofluorescence images of RPA32 foci (red) and 53BP1 foci (green) in control and XAB2-depleted U87 cells exposed to 15 µM TMZ (or DMSO) for 2 h and allowed to recover in drug-free medium for 48 h (B), and related quantification of the average number of RPA32 foci per nucleus (C). (D) Quantification of the average number of RAD51 foci in control and XAB2-depleted cells exposed to 15 µM TMZ (or DMSO) for 2 h and allowed to recover in drug-free medium for the indicated times. See Supplementary Figure S2 for representative immunofluorescence images. (E and F) Quantification of the average number of pS1778-53BP1 foci per nucleus, in the total cell population, as well as in Cyclin A-negative and -positive cell subpopulations, in control and XAB2-depleted U87 cells following exposure to 15 µM TMZ (or DMSO) for 2 h, 72 h recovery in drug-free medium and processing for IF analysis of pS1778-53BP1 foci and Cyclin A (E), and representative immunofluorescence images (F). Scale bar: 5µm. The images are representative of three or more independent biological repeats. Bars represent mean ± s.e.m. Significant differences between specified comparisons were assessed by two-ways ANOVA and are highlighted by stars (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001).

Fig 4: DNA-damage-induced RPA phosphorylation promotes its ubiquitylation. (A) HeLa cell lines stably expressing HA-tagged WT or Ala10 RPA32 mutants were transfected with a vector expressing SFB-PRP19. Cells were then treated with CPT 1 µM for 4 h, lysed and SFB-PRP19 and its interactors were isolated using streptavidin-associated beads. (B) Stable HeLa cell lines expressing the indicated HA-tagged RPA32 constructs were transfected with an siRNA targeting the 3' untranslated region for the RPA32 mRNA. 72 h later, cells were treated with 1 µM CPT for 2 h, lysed and the indicated proteins were detected using specific antibodies. (C) Alternatively, cells transfected as in (B) were microirradiated and processed for immunofluorescence to examine PRP19 recruitment to laser stripes. (D) Histogram representing the recruitment of endogenous PRP19 to RPA32 stripes after laser microirradiation. The error bars correspond to biological triplicate experiments. At least 100 microirradiated cells were examined for each replicate. (E) Cells were transfected with vectors expressing His6-tagged ubiquitin and myc-tagged WT or Ala10 RPA32 mutants and treated with 1 µM CPT for 3 h. Ubiquitylated proteins were isolated by denaturing Ni-NTA pulldown.

Fig 5: HR factors are recruited, and damage-induced DNA synthesis can be observed at I-PpoI-induced nucleolar caps in G1 cells. (A) I-PpoI transfection of RPE1 cells stably expressing FUCCI component mCherry-Cdt1 reveals that HR factors Rad51 and RPA2 are recruited comparably to nucleolar caps in G1 (red) and late S/G2 (nonred) cells. Quantification is shown below. (B) I-PpoI transfection of RPE1 cells stably expressing FUCCI component mAG-Geminin reveals that HR factors Rad51 and RPA2 are recruited comparably to nucleolar caps in G1 (nongreen) and late S/G2 (green) cells. Quantification is shown below. (C) RPE1 cells stably expressing FUCCI component mAG-Geminin or mCherry-Cdt1 were transfected with I-PpoI mRNA; 4 h after transfection, fresh medium containing EdU was added to cells. Following a 2-h pulse of EdU labeling, cells were fixed. Treacle antibodies detected damage-induced nucleolar caps. Damage-induced DNA synthesis was visualized using biotin-azide and fluorophore-conjugated streptavidin (see the Materials and Methods for details). In the top right panel, EdU incorporation can be observed at damage-induced nucleolar caps in G1 (nongreen) mAG-Geminin cells. In the middle panels, EdU incorporation is observed in nucleolar caps in both G1 (red) and G2 (nonred) mCherry-Cdt1 cells. A DIC image is shown at the right. In the bottom panels, a single enlarged G1 cell from the same experiment is shown.