Fig 2: rDNA resection occurs downstream of ATR activation.a Timecourse of RPA2 foci formation in U2OS cells after I-Ppo1 transfection. b Timecourse of RPA2 foci formation in cells counterstained for Cyclin A. c Quantification of the experiment in b (all data points shown, red bars represent mean). d Timecourse of RPA2 foci formation in U2OS WT and NBS1?N cells after I-Ppo1 transfection. e Quantification of the experiment in d (all data points shown, red bars represent mean). f Timecourse of RPA2 foci formation in U2OS cells treated with ATMi (KU-55933) and ATRi (VE-821) after I-Ppo1 transfection. g Quantification of the experiment in f (all data points shown, red bars represent mean). All scalebars = 10 µm. Source data are provided as a Source Data file.

Fig 3: Loss of Treacle and TOPBP1 impairs rDNA repair.a ?H2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and Treacle-depleted (siTreacle) U2OS cells 2 h after I-Ppo1 transfection. b Quantification of the experiment in a, ?H2AX: siCtrl (n = 177) versus siTreacle (n = 200), 53BP1: siCtrl (n = 261) versus siTreacle (n = 239), RPA2: siCtrl (n = 152) versus siTreacle (n = 168) BRCA1: siCtrl (n = 525) versus siTreacle (n = 246) and RAD51: siCtrl (n = 525) versus siTreacle (n = 246) cells (all datapoints are shown, bar represents mean, error bars represent S.D.) c ?H2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and TOPBP1-depleted (siTOPBP1) U2OS cells 2 h after I-Ppo1 transfection. d Quantification of the experiment in c, ?H2AX: siCtrl (n = 403) versus siTOPBP1 (n = 205), 53BP1: siCtrl (n = 239) versus siTOPBP1 (n = 256), RPA2: siCtrl (n = 649) versus siTOPBP1 (n = 359) BRCA1: siCtrl (n = 228) versus siTOPBP1 (n = 348) and RAD51: siCtrl (n = 228) versus siTOPBP1 (n = 348) cells (all datapoints are shown, bar represents mean, error bars represent S.D.). e Clonogenic surivival analysis of I-Ppo1-transfected control (siCtrl), Treacle-depleted (siTreacle) and TOPBP1-depleted (siTOPBP) U2OS cells (red bars represent the mean). f Clonogenic surivival analysis of I-Ppo1-transfected WT and NBS1DN U2OS cells (red bars represent the mean). g Model (see text for details). Source data are provided as a Source Data file.

Fig 4: RPA2 is a PCAT19 interaction partner(A) Biotin-tagged antisense-oligo (AS-oligo) RNA pull-down of PCAT19 and its interacting proteins from endothelial cell lysate measured by mass spectrometry. Scrambled AS-oligos were used as negative control (CTL). A log10iBAQ representation of enriched proteins (sum of all peptide intensities/number of observable peptides) against the log2 fold difference of PCAT19/CTL is shown (n = 6). Highlighted proteins indicate enrichment with PCAT19 (p < 0.05; q < 0.05). iBAQ, intensity-based absolute quantitation.(B) Table of significantly enriched PCAT19-interacting proteins (p < 0.05, q < 0.05).(C) Schematic depicting the proteins pulled down with PCAT19 and potential interaction map based on literature searches. RPA2 is central to the RPA complex and reportedly functions alongside most of the proteins identified with mass spectrometry.(D) AS-oligo RNA pull-down of PCAT19 (P19) or control AS-oligos (CTL) and western blot with antibodies against RPA2 and PKNP. Lamin B1 served as negative control.(E) RNA immunoprecipitation (RIP) in HUVEC extract with an antibody against RPA2 followed by qRT-PCR for PCAT19. Percentage of input recovery of PCAT19 versus a non-primary-antibody control (IgG) is shown. 18S rRNA and U4 snRNA served as negative controls.(F) In vitro binding assay of RPA2 and PCAT19. His-tagged RPA2 was combined with in vitro-transcribed and biotinylated PCAT19 or pcDNA3.1+ control RNA (biotin-CTL). Streptavidin beads were used to pull down the biotin-tagged RNAs and blots stained for RPA2. Data are presented as the mean ± SD; **p < 0.01.

Fig 5: PCAT19 limits RPA2 serine 33 (S33) phosphorylation(A) Depiction of RPA2 phosphorylation sites.(B) HUVECs were seeded at subconfluent or confluent levels and treated with 10 µM ATRi or DMSO for 16 h. Western blot staining for RPA2 and S33-pRPA2 is shown (n = 3).(C) HUVECs were transfected with LNA GapmeRs against PCAT19 or negative control LNA and treated with 10 µM ATRi or DMSO for 16 h. Western blot staining for RPA2 and S33-pRPA2 is shown (n = 3).(D) HUVECs were transduced with either PCAT19 overexpression (OE) plasmid or pcDNA3.1+ backbone control plasmid and then treated with 10 µM ATRi or DMSO for 16 h. Western blot staining for RPA2 and S33-pRPA2 is shown (n = 6).(E) In vitro binding assay for various combinations of His-RPA2, biotin-PCAT19, biotin-CTL RNA, ATRi, and phosphatase. Staining of S33-pRPA2 or RPA2 in biotin pull-down and 5% input samples is shown.(F) In vitro phosphorylation assay of endogenous RPA2 and recombinant His-RPA2. Combinations of His-RPA2, FLAG-ATR, in vitro-transcribed PCAT19, ATR inhibitor, phosphatase (CIP), phosphatase inhibitor, and ATP are shown. FLAG, His, and S4/8-pRPA2 antibodies were used for staining.(G) HUVECs were transduced with either PCAT19 overexpression (OE) plasmid or pcDNA3.1+ backbone control plasmid. Duolink proximity ligation assay for RPA2-ATR is shown. Red signal indicates the Duolink PLA signal (546 nm), blue indicates DAPI.(H) EdU proliferation assay after PCAT19 LNA-GapmeR-mediated knockdown or control LNA and with or without treatment with 10 µM ATRi for 16 h (n = 3).(I) Spheroid outgrowth assay after PCAT19 LNA-GapmeR-mediated knockdown or control LNA and with or without treatment with 10 µM ATRi for 16 h (n = 3). Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.