1. Volume 22, Issue 2, Pages 383-395 (January 2018)
RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair 
Mohammad A.M. Ali, Hilmar Strickfaden, Brian L. Lee, Leo Spyracopoulos, Michael J. Hendzel 
Cell Reports 
Volume 22, Issue 2, Pages (January 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 383-395DOI: (10.1016/j.celrep.2017.12.047)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
RYBP Preferentially Binds K63 Diubiquitin and Is Displaced from Sites of DSBs
(A) GST pull-down of mono- and di-Ub by GST-tagged RYBP-NZF. Binding was monitored by visualizing the decrease in free ubiquitin remaining in the supernatant after pull-down with increasing amounts of NZF domain using an SDS-PAGE Coomassie-blue-stained gel.
(B) Representative regions of 2D 1H-15N HSQC spectra of 15N-Ub titrated with increasing amounts of NZF (left) and 15N-NZF titrated with Ub (right) showing the chemical shift changes of the respective proteins on increasing titrant concentration. To the right of each spectra are surface representations of Ub (PDB: 1UBQ) and a model of the RYBP-NZF domain based on the YAF2-NZF domain (PDB: 2DG9). Residues are colored from white to red based on the magnitude of the chemical shift changes in the NMR titrations and show the Ub-NZF binding interface. See also Table S1.
(C) U2OS cells tested for co-localization of RYBP with DSBs. Top left: distribution of GFP-RYBP within the nucleus. Top right: DNA-damage foci after IR illustrated by anti-γH2AX. Bottom left: DAPI stain. Bottom right: merged GFP-RYBP and γH2AX channels. Scale bar represents 10 μm.
(D) Kinetics of displacement of RYBP from DNA damage sites. Arrows indicate the area of laser microirradiation. Scale bar represents 10 μm.
(E) Quantification of GFP-RYBP displacement (n = 21) from three independent experiments. Error bars represent SEM.
(F) U2OS-DSB reporter cells stably expressing mCherry-LacI-FokI nuclease. DSBs were induced, and ChIP was performed with indicated antibodies. qPCR was performed using two primers, and the DSB-induced enrichment for each primer and antibody is plotted. Error bars represent SEM for two independent experiments.
See also Figure S1.
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4. Figure 2
RYBP Displacement from DSB Sites Is Ubiquitylation and p97 Dependent
(A) GFP-RYBP displacement in the presence of vehicle control or two transcription inhibitors (DRB and cordycepin). Right: quantification of the displacement (n = 15) from two independent experiments.
(B) GFP-RYBP displacement in RNF8+/+ or RNF8−/− MEFs. Right: quantification of the displacement of GFP-RYBP (n = 21–30) from three independent experiments.
(C) GFP-RYBP displacement in cells treated with proteasome inhibitor (MG132), selective E1 ubiquitin-activating enzyme inhibitor (PYR41), or specific p97 inhibitor (DBeQ). Right: quantification of the displacement (n = 21) from three independent experiments.
(D) GFP-RYBP displacement upon p97 knockdown using two siRNAs (si-p97 1 and 2) in comparison to negative control. Right: quantification of the displacement (n = 30) from three independent experiments.
Scale bars represent 10 μm. Arrows indicate the area of microirradiation. Error bars represent SEM. See also Figure S2.
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5. Figure 3 K48 Polyubiquitylation of RYBP upon DNA Damage
(A) Immunoblot of GFP-RYBP immunoprecipitated by GFP-Trap (ChromoTek) with or without IR using anti-GFP (top) or linkage-specific anti-ubiquitin (middle) antibodies. Immunoblot of γH2AX in corresponding cell lysates (bottom).
(B) Top: immunoblot of GFP-RYBP immunoprecipitated using anti-ubiquitin TUBE1 agarose (LifeSensors) with or without IR. Increased signal after DUB treatment (USP2) indicates polyubiquitylation. Bottom: quantification of the signal intensities from three independent experiments.
(C) Top: in vitro ubiquitylation of GFP-RYBP upon incubation with RNF8. Arrowheads indicate ubiquitylated products. Bottom: GFP only control shows no ubiquitylation.
(D) Top: U2OS cells co-express mCherry-RYBP and GFP-ubiquitin mutants and subjected to microirradiation. (Ub-WT, wild-type ubiquitin; Ub-noK, all K residues mutated to R; Ub-K48, all K mutated to R except K48; Ub-K48R, only K48 residue mutated to R). Bottom: quantification of displacement of mCherry-RYBP at 180 s post microirradiation from three independent experiments. Scale bars represent 10 μm. Arrows indicate the area of microirradiation.
Error bars represent SEM; ∗p < See also Figure S3.
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6. Figure 4
The RYBP Ubiquitin-Binding Motif Is Required for Its Removal from DNA Damage Sites
(A) Schematic diagram of RYBP mutant constructs.
(B) GFP-RYBP wild-type and mutants illustrated in (A) were monitored in living cells by time-lapse microscopy following laser microirradiation.
(C) Quantification of the displacement or recruitment of GFP-RYBP mutants (n = 21) from three independent experiments. Scale bars represent 10 μm. Arrows indicate the area of microirradiation. Error bars represent SEM.
(D) GFP-RYBP-WT, RYBP-TF-AA and -ΔNZF mutants immunoprecipitated with K63-ubiquitin conjugated beads, then analyzed by western blotting using an anti-GFP antibody.
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7. Figure 5 RYBP Reduces Recruitment of DDR Proteins to IRIF
(A) Immunofluorescence of GFP-RYBP, RAP80, and γH2AX in U2OS cells fixed 1 hr after IR (5 Gy).
(B) Right: GFP-RAP80 recruitment to DNA damage sites induced by laser microirradiation in U2OS cells that co-express either mCherry or mCherry-RYBP. Left: quantification of the recruitment of GFP-RAP80 (n = 10) from two independent experiments. Scale bars represent 10 μm. Arrows indicate the area of microirradiation, and error bars represent SEM.
(C) Immunofluorescence of GFP-RYBP, BRCA1, and γH2AX in U2OS cells fixed 1 hr after IR (2 Gy).
(D) Immunofluorescence of GFP-RYBP, Rad51, and γH2AX in U2OS cells fixed 3 hr after IR (2 Gy).
(E) Immunofluorescence of GFP-RYBP, 53BP1, and γH2AX in U2OS cells 1 hr after IR (2 Gy).
(F) Immunofluorescence of GFP-RYBP-TF-AA mutant, BRCA1, and γH2AX in U2OS cells transfected with GFP-RYBP-TF-AA mutant and fixed 1 hr after IR (2 Gy).
In (A) and (C)–(F) (right), quantification of the average indicated focus intensity in non-transfected versus RYBP expressing cells is shown (n = 50, three independent experiments). See also Figure S4.
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8. Figure 6 Effect of RYBP on HR Repair and Sensitivity to IR
(A) Immunofluorescence of GFP-RYBP and RPA in cyclin A-positive cells fixed 3 hr after IR (2 Gy).
(B) Immunofluorescence of GFP-RYBP-TF-AA mutant and RPA in cyclin-A-positive cells fixed 3 hr after IR (2 Gy).
In (A) and (B) (right), quantification of the average RPA focus intensity in non-transfected versus indicated RYBP expressing cells is shown (n = 30, three independent experiments).
(C) Immunoblot of phosphorylated RPA2, total RPA, and γH2AX 6 hr after IR (6 Gy) in U2OS cells that stably express GFP, GFP-RYBP, or GFP-RYBP-TF-AA mutant.
(D) Single-stranded DNA was detected by BrdU detection in cyclin-A-positive HeLa cells transfected with GFP-RYBP without DNA denaturation in cells fixed 3 hr after IR exposure.
(E) HR-mediated gene conversion assay in TRI-DR-GFP U2OS cells expressing mCherry-RYBP. HR efficiencies are normalized to controls expressing mCherry. Error bars represent SD from three independent experiments. See also Figure S5.
(F) Control U2OS cells were mixed equally with U2OS stably express either GFP or GFP-RYBP and subjected to increasing doses of radiation. The percentage of GFP positive cells was assessed 7 days post-IR.
(G) Clonogenic survival of U2OS cells stably expressing GFP, GFP- RYBP, RYBP-ΔC, or RYBP-TF-AA and subjected to the indicated doses of IR.
(H) Clonogenic survival of U2OS cells upon RYBP knockdown relative to negative control.
In (G) and (H), error bars represent SEM from three independent experiments; ∗p < 0.05.
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9. Figure 7
Endogenous RYBP Levels Are Associated with Radiosensitivity in Breast Cancer Cell Lines
(A) Immunoblot of endogenous RYBP in the indicated breast cancer cell lines. Right: quantification of RYBP relative to actin from three independent experiments. See also Table S2.
(B) Immunoblot of RYBP in U2OS that stably express GFP-RYPB or in MDA-MB-453. Right: quantification of GFP- or endogenous RYBP relative to actin in U2OS cells or MDA-MB-453, respectively.
(C) Immunofluorescence staining of BRCA1 and γH2AX in the indicated breast cancer cells subjected to IR (2 Gy) fixed 1 hr later. Bottom: quantification of the average BRCA1 focus intensity (n = 50 from three independent experiments). See also Figure S6.
(D) Immunofluorescence staining of BRCA1 and RYBP in MDA-MB-453 after RYBP knockdown. Cells were subjected to IR (2 Gy) and fixed 1 hr later. Bottom: quantification of the average BRCA1 focus intensity (n = 50 from three independent experiments).
(E) Clonogenic survivals of MDA-MB-231, MDA-MB-468, or MDA-MB-453 cells upon RYBP knockdown relative to negative control with indicated doses of IR. Error bars indicate SEM from three independent experiments.
∗p < See also Figure S7.
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