KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
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| Antibodies | ||
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| Rabbit polyclonal anti-BRCA1 | Cell Signaling Technology | Cat# 9010; RRID: AB_2228244 |
| Rabbit polyclonal anti-FANCJ (E67) | Cantor Laboratory | N/A |
| Mouse monoclonal anti-β-actin, clone AC-15 | Sigma-Aldrich | Cat# A5441; RRID: AB_476744 |
| Mouse monoclonal anti-BrdU (B44) | BD Biosciences | Cat# 347580; RRID: AB_400326 |
| Rat monoclonal anti-BrdU, clone BU1 / 75 (ICR1) | Abcam | Cat# ab6326; RRID: AB_305426 |
| Rabbit monoclonal anti-p21 Waf1/Cip1 (12D1) | Cell Signaling Technology | Cat# 2947; RRID: AB_823586 |
| Rabbit polyclonal anti-53BP1 | Novus Biological | Cat# NB100-304; RRID: AB_10003037 |
| Mouse monoclonal anti-CHD4 [3F2/4] | Abcam | Cat# ab70469; RRID: AB_2229454 |
| Rabbit polyclonal anti-BRCA2 | Abcam | Cat# ab123491; RRID: AB_10972163 |
| Rabbit polyclonal anti-CXorf57 (RADX) | Abcam | Cat# ab228707 |
| Mouse monoclonal anti-PCNA [PC10] | Abcam | Cat# ab29; RRID: AB_303394 |
| Rabbit monoclonal anti-RPA70 [EPR3472] | Abcam | Cat# ab79398; RRID: AB_1603759 |
| Rabbit monoclonal anti-MCM6 [EPR17686] (Alexa Fluor® 568) | Abcam | Cat# ab211916 |
| Mouse monoclonal anti-PCNA (PC10) | Santa Cruz Biotechnology | Cat# sc-56, RRID: AB_628110 |
| Rabbit polyclonal anti-RPA70/RPA1 | Cell Signaling Technology | Cat# 2267; RRID: AB_2180506 |
| Mouse monoclonal anti-γH2AX (Ser139), clone JBW301 | Millipore | Cat# 05-636; RRID: AB_309864 |
| Mouse monoclonal anti-RPA32/RPA2 [9H8] | Abcam | Cat# ab2175; RRID: AB_302873 |
| Mouse monoclonal anti-poly(ADP-ribose) | Trevigen | Cat# 4335-MC-100; RRID: AB_2572318 |
| Rabbit monoclonal anti-FEN1[EPR4460(2)] | Abcam | Cat# ab109132; RRID: AB_10866125 |
| Rabbit polyclonal anti-PARP1 | Abcam | Cat# ab227244 |
| Rabbit monoclonal anti-XRCC1 [EPR4389(2)] |
Abcam | Cat# ab134056 |
| Mouse monoclonal anti-DNA ligase III [1F3] | GeneTex | Cat# GTX70143; RRID: AB_372134 |
| Rabbit polyclonal anti-Histone H2B (V119) | Cell Signaling Technology | Cat# 8135; RRID: AB_10891053 |
| Mouse monoclonal anti-DNA Ligase I (C-5) | Santa Cruz Biotechnology | Cat# sc-271678; RRID: AB_10708845 |
| Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) | Cell Signaling Technology | Cat# 9661; RRID: AB_2341188 |
| Rabbit polyclonal anti-PARP (cleaved PARP1) | Cell Signaling Technology | Cat# 9542; RRID: AB_2160739 |
| ECL anti-rabbit IgG, HRP-linked whole antibody (from donkey) | GE Healthcare | Cat# NA934V; RRID: AB_772206 |
| Rb anti-Ms IgG secondary antibody, HRP conjugate | Thermo Fisher Scientific | Cat# 61-6520; RRID: AB_2533933 |
| Alexa Fluor 488 goat anti-Mouse IgG (H+L) | Thermo Fisher Scientific | Cat# A-11001; RRID: AB_2534069 |
| Alexa Fluor 594 goat anti-Rat IgG (H+L) | Thermo Fisher Scientific | Cat# A-11007; RRID: AB_141374 |
| Alexa Fluor 568 goat anti-Rabbit IgG (H+L) | Thermo Fisher Scientific | Cat# A-11011; RRID: AB_143157 |
| Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) | Invitrogen | Cat# A-11029; RRID: AB_138404 |
| Alexa Fluor 750 Goat Anti-Rabbit IgG (H+L) | Invitrogen | Cat# A-21039; RRID: AB_10375716 |
| Alexa Fluor 555 goat anti-Rabbit IgG (H+L) | Thermo Fisher Scientific | Cat# A-21428; RRID: AB_141784 |
| Rhodamine Red-X-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch Labs | Cat# 115-295-062; RRID: AB_2338761 |
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| Biological samples | ||
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| Patient-derived xenograft (PDX) PNX017 | This paper | N/A |
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| Chemicals, peptides, and recombinant proteins | ||
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| IdU (5-Iodo-2′-deoxyuridine) | Sigma-Aldrich | Cat# I7125 |
| CldU (5-chloro-2′-deoxyuridine) | Sigma-Aldrich | Cat# C6891 |
| Olaparib | SelleckChem | Cat# AZD2281 |
| Cisplatin | Sigma-Aldrich | Cat# P4394 |
| Camptothecin (CPT) | Sigma-Aldrich | Cat# C9911 |
| Mitomycin C (MMC) | Sigma-Aldrich | Cat# M4287 |
| S1 nuclease | Thermo Fisher Scientific | Cat# 18001-016 |
| VE-821 (ATR inhibitor) | SelleckChem | Cat# S8007 |
| Hydroxyurea | Sigma-Aldrich | Cat# H8627 |
| RPA inhibitor | NERx Biosciences | Cat# NERx-329 |
| PDD 00017273 (PARG inhibitor) | Tocris | Cat# 5952 |
| Emetine | MedChemExpress | Cat# HY-B1479B |
| Methyl methansulfonate (MMS) | Sigma-Aldrich | Cat# 129925 |
| LNT1 (FEN1 inhibitor) | Tocris | Cat# 6510 |
| L189 (Ligase I/II/IV inhibitor) | MedChemExpress | Cat# HY-15588 |
| Z-VAD-FMK (pan-caspase inhibitor) | SelleckChem | Cat# S7023 |
| Emricasan (pan-caspase inhibitor) | SelleckChem | Cat# S7775 |
| DMSO (Dimethyl sulfoxide) | Sigma-Aldrich | Cat# D5879 |
| Puromycin | Sigma-Aldrich | Cat# P8833 |
| Crystal violet | Sigma-Aldrich | Cat# C0775 |
| Formaldehyde | Fisher Chemical | Cat# F79-500 |
| EdU (5-ethynyl-2′-deoxyuridine) | Thermo Fisher Scientific | Cat# A10044 |
| Paraformaldehyde | Electron Microscopy Sciences | Cat# 15714 |
| Paraformaldehyde | Sigma-Aldrich | Cat# P6148 |
| Acetone | Fisher Chemical | Cat# A18 |
| Methanol | Fisher Chemical | Cat# A412 |
| DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | Cat# D1306 |
| ProLong Gold Antifade reagent | Thermo Fisher Scientific | Cat# P36930 |
| VECTASHIELD mounting media | Vector Laboratories | Cat# H-1200 |
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| Critical commercial assays | ||
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| Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 | Invitrogen | Cat# C10337 |
| CellTiter-Glo 2.0 Assay | Promega | Cat# G9242 |
| Click-iT Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye | Thermo Fisher Scientific | Cat# C10640 |
| Cell Counting Kit-8 (CCK-8) | Dojindo | Cat# CK04 |
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| Experimental models: cell lines | ||
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| Human: RPE1-hTERT TP53−/− | Durocher Laboratory | https://pubmed.ncbi.nlm.nih.gov/30022168/ |
| Human: RPE1-hTERT TP53−/−;BRCA1−/− | Durocher Laboratory | https://pubmed.ncbi.nlm.nih.gov/30022168/ |
| Human: RPE1-hTERT TP53−/−;BRCA1−/−;TP53BP1−/− |
Durocher Laboratory | https://pubmed.ncbi.nlm.nih.gov/30022168/ |
| Human: RPE1-hTERT TP53−/−;FANCJ−/− | This paper | N/A |
| Human: U2OS | Cantor Laboratory | https://pubmed.ncbi.nlm.nih.gov/30232006/ |
| Human: U2OS FANCJ−/− | Cantor Laboratory | https://pubmed.ncbi.nlm.nih.gov/30232006/ |
| Human: 293T | Cantor Laboratory | https://pubmed.ncbi.nlm.nih.gov/30232006/ |
| Human: 293T FANCJ−/− | Cantor Laboratory | https://pubmed.ncbi.nlm.nih.gov/30232006/ |
| Mouse: T2 | Zou Laboratory | https://pubmed.ncbi.nlm.nih.gov/28242626/ |
| Mouse: BR5 | Zou Laboratory | https://pubmed.ncbi.nlm.nih.gov/28242626/ |
| Mouse: BR5-R1 | Zou Laboratory | https://pubmed.ncbi.nlm.nih.gov/28242626/ |
| Human: PEO1 | Taniguchi Laboratory | https://pubmed.ncbi.nlm.nih.gov/19654294/ |
| Human: C4-2 | Taniguchi Laboratory | N/A |
| Hamster: V-C8 | Jasin Laboratory | https://pubmed.ncbi.nlm.nih.gov/21565612/ |
| Hamster: V-C8+BRCA2 | Jasin Laboratory | https://pubmed.ncbi.nlm.nih.gov/21565612/ |
| Hamster: V-C8+BRCA2 S3291A | Jasin Laboratory | https://pubmed.ncbi.nlm.nih.gov/21565612/ |
| Human: FA Patient fibroblasts RA2630 T131P |
Smogorzewska Laboratory | https://pubmed.ncbi.nlm.nih.gov/26253028/ |
| Human: FA Patient fibroblasts CRISPR corrected clone 3–39 ΔMUT/WT or -/WT | Smogorzewska Laboratory | https://pubmed.ncbi.nlm.nih.gov/26253028/ |
| Human: FA Patient fibroblasts CRISPR double-allele corrected clone | Smogorzewska Laboratory | Available by request |
| Human: UWB1 | Turchi Laboratory | N/A |
| Human: UWB1+BRCA1 | Turchi Laboratory | N/A |
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| Experimental models: organisms/strains | ||
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| Mouse: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ | Johnson Laboratory | https://pubmed.ncbi.nlm.nih.gov/30257212/ |
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| Oligonucleotides | ||
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| FANCJ CRISPR K/O gRNA | This paper | Table S2 |
| shRNA sequences | This paper | Table S2 |
| siRNA scrambled negative control | ORIGENE | Cat# SR30004 |
| siRNA FANCJ/BRIP1 | QIAGEN | Cat# SI03110723 |
| siRNA BRCA1 | QIAGEN | Cat# SI00299495 |
| siRNA RPA1 | Dharmacon | Cat# D-015749-04-0005 |
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| Recombinant DNA | ||
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| Plasmid: AcGFP-RPA3-P2A-RPA1-P2A-RPA2 fusion construct | (Toledo et al., 2013) | N/A |
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| Software and algorithms | ||
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| Prism 7.0 | GraphPad | https://www.graphpad.com/scientificsoftware/prism/ |
| CellProfiler-3.1.5 | Broad Institute | https://cellprofiler.org/releases |
| Fiji (ImageJ) | NIH | https://imagej.nih.gov/ij/docs/guide/146-2.html |
| ZEN blue 2.5 pro | Zeiss | N/A |
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| Deposited data | ||
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| Raw and uncropped data | This paper; Mendeley | https://doi.org/10.17632/g96fktpb58.1 |
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| Other | ||
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| Lipofectamine RNAiMAX transfection reagent | Invitrogen | Cat# 13778150 and P/N 56532 |
| X-tremeGENE HP DNA Transfection Reagent | Roche | Cat# 6366244001 |
| Genomic DNA Extraction Kit | Genomic Vision | Cat# EXT-001 |
| β-agarase I | New England Biolabs | Cat# M0392 |