Fig 2: The ATR kinase facilitates repression of nucleolar transcription and nucleolar cap formation. (A) Measurements of transcription by 5-EU incorporation in MRE11-depleted U2OS-Cas9-NBS1-GFP cells treated with gRNAs at indicated time points. Prior to fixation cells were labeled with 5-EU and samples were subsequently processed with Click-iT chemistry. (B) Analysis of NBS1-GFP nucleolar foci after ATR inhibition. U2OS-Cas9-NBS1-GFP cells were treated with the indicated ATR inhibitor and transfected with gRNAs. Samples were harvested at indicated time points and immunostained with antibodies against MRE11. Zoomed images show details of individual nucleoli with dotted lines marking the edge of the nucleoli. (C) Count of cells with NBS1-GFP interior nucleolar foci from experiments carried out as in b using three ATR inhibitors. Error bars represent SD, n = 3, multiple comparisons of the mean for all time points were carried out with unpaired t-tests, and the two-sided P-values were adjusted using the Holm–Sidak method. Statistical significance is depicted with stars (* = 0.05–0.01, ** = 0.01–0.001, *** = 0.001–0.0001, **** = 0.0001) and no legend = ns. (D) Temporal behaviour of NBS1-GFP foci up to 10 h after gRNA transfection combined with ATR inhibition (VE821) analysed using timelapse microscopy. Insets show enlarged individual nucleoli. (E) Measurements of transcription by 5-EU incorporation in ATR inhibitor and gRNA treated U2OS-Cas9-NBS1-GFP cells. Prior to fixation cells were labeled with 5-EU and samples were subsequently processed with Click-iT chemistry. (F) Recruitment of repair proteins after ATR inhibition. Staining patterns of NBS1-GFP, BRCA1 and 53BP1 were analysed by immunostaining with antibodies against BRCA1 and 53BP1 in cells treated with ATR inhibitor and gRNA.

Fig 3: Ectopic BRCA1 expression rescues SPT6 loss phenotype.a Representative immunoblot analysis of BRCA1, SPT6, RAD51, total and phosphorylated ATM expression in U2OS-GFP and U2OS-BRCA1-GFP cells transfected with siCON or siSPT6-p (pool). Loading control: a-Tubulin and GAPDH. b ?H2AX foci quantification in U2OS-GFP and U2OS-BRCA1-GFP cells transfected with either siCON or siSPT6-p. Data are presented as mean ± s.d. *p = 0.0331; one-way ANOVA followed by Tukey’s multiple comparison test. c Representative DSBs quantification using comet assay. Data are presented as mean ± s.d. ****p < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test. Representative images are shown on left. d 53BP1 foci quantification in G1 and S/G2 phase U2OS-GFP and U2OS BRCA1-GFP cells transfected with siCON or siSPT6-p. Non-significant ns = 0.8471 and ****p < 0.0001; two-way ANOVA followed by Tukey’s multiple comparison test. N = 3 biologically independent experiments in (a–d). Source data are provided as a Source data file.

Fig 4: SIRT2 deacetylase activity promotes BRCA1-BARD1 stability(A–C) SIRT2 knockdown decreases BRCA1 and BARD1 protein levels but does not significantly change mRNA levels in HCT116 cells.(D–F) AGK2 treatment for 24 h decreases BRCA1 and BARD1 protein levels but shows no significant change in mRNA levels in HCT116 cells.(G and H) HEK293T cells were treated with cycloheximide with or without AGK2 for 0, 2, or 4 h. AGK2 increases the rate of BRCA1 degradation in HEK293T cells compared with a control. Mean and SD from four independent replicas are shown.(I) Treatment with MG132 for 6 h alleviates BRCA1 levels after SIRT2 knockdown in HCT116 cells.(J) Treatment with MG132 for 6 h alleviates BARD1 levels after SIRT2 knockdown in HCT116 cells.For (C) and (F), mean and SD from three independent replicas are shown. For (D) and (E), acetylated a-tubulin acts as a positive control for AGK2 treatment. *p < 0.05.See also Figure S2.

Fig 5: Localization of HR repair factors during lytic replication of KSHV. (A) EA.hy926-RTA cells containing latent KSHV were treated with doxycycline for 48 h or left untreated prior to IF analysis of nuclear localization of BRCA1 in relation to KSHV RCs marked by the viral SSB protein. Latently infected U2OS cells expressing GFP-CtIP were treated with sodium butyrate for 48 h or left untreated prior to IF analysis of GFP-CtIP localization in relation to KSHV RCs marked by the viral SSB protein. (B) EA.hy926-RTA cells were prepared as described above prior to IF analysis of nuclear localization of the HR repair factors RAD51, RAD52, and RAD54B in relation to KSHV RCs marked by the viral PF8 protein. Scale bars represent 5 µm.