Fig 1: RAD52 is required for HR when long nonhomologous tails or G4s are present at the DSB ends after I-SceI cleavage.A, schematic drawing of the HR-EGFP reporter containing a nonhomologous sequence (orange) in the recipient cassette serving as a nonhomologous tail after I-SceI cleavage (top). Models of HR repair at DSB ends containing nonhomologous tails (bottom). XPF/ERCC1 cleaves nonhomologous tails after strand invasion (a, left) or after second-end capture (b, right). B, U2OS cells carrying the HR-EGFP (Luc-390bp) reporter were depleted for XPF, RAD52, or both XPF and RAD52 by shRNAs with vector as control. Relative HR frequency was determined by FACS analysis 4 days after infection with lentiviruses producing I-SceI. C, U2OS cells carrying the HR-EGFP reporters with different lengths of inserted luciferase sequences (Luc-200 bp, Luc-40 bp, and Luc-13 bp) were depleted for XPF or RAD52 by shRNAs with vector as control. Relative HR frequency was determined by FACS analysis 4 days after the lentiviral infection of I-SceI. D, schematic drawing of the HR-EGFP (TPG4) reporter with a 40 bp insertion containing the TPG4 sequence with the I-SceI cleavage sites indicated (top). Upon I-SceI cleavage, G4 structures would form on the ssDNA overhangs after end resection (bottom). E, U2OS cells carrying the HR-EGFP (TPG4) reporter were depleted for XPF, RAD52, or both XPF and RAD52 by shRNAs with vector as control. Relative HR frequency was determined by FACS analysis 4 days after the lentiviral infection of I-SceI. F, RAD52 WT allele or R55A mutant allele was expressed in U2OS [HR-EGFP (TPG4)] cells (left) and U2OS [HR-EGFP (Luc-390 bp)] cells (right) and the endogenous RAD52 was depleted by shRNA (the shRNA targeting site in RAD52-WT and R55A was mutated). The relative HR frequency was determined by FACS analysis 4 days after the lentiviral infection of I-SceI. In all experiments, error bars represent the SD of at least three independent experiments. DSB, double-stranded break; EGFP, enhanced GFP; FACS, fluorescence activated cell sorting; G4, G-quadruplexes; HR, homologous recombination.
Fig 2: RAD52 is synthetically lethal with FANCJ.A, U2OS cells were depleted for RAD52, FANCJ, or both by shRNAs with a vector control. γ-H2AX Western blot analysis was performed before and after PDS treatment (50 μM, 24 h) using GAPDH as the loading control (left). Depletion efficiency was determined by Western blot analysis using indicated antibodies (right). B, U2OS WT and FANCJ KO cells were infected with RAD52 shRNA lentiviruses or vector control, and cell growth curves were plotted (left). Error bars represent the SD of at least three independent experiments. RAD52 depletion and FANCJ KO were verified by Western blot analysis using indicated antibodies (right). C, U2OS WT and MUS81KO cells were infected with FANCJ shRNA (left) or RAD52 shRNA (right) lentiviruses or vector control, and cell growth curves were plotted (top). Error bars represent the SD of at least three independent experiments. Depletion of FANCJ and RAD52 was verified by Western blot analysis using indicated antibodies (bottom). PDS, pyridostatin.
Fig 3: RAD52 deficiency causes increased DSB formation and cell death after the treatment of G4-stabilizing drugs.A, U2OS and U2OS-derived RAD52 KO cells were treated with the indicated concentrations of PDS (left) and CX-5461 (middle) for 48 hours (h), and cell viability assays were performed. RAD52 Western blot was performed to show RAD52 KO with KU70 as a loading control (right). B, EGFP-RAD52 was expressed in U2OS cells and representative EGFP-RAD52 foci are shown with DAPI staining before and after PDS treatment (50 μM, 48 h, left). EGFP-RAD52 foci/nucleus in PDS-treated and -untreated cells were qualified and plotted (right). The p value is indicated as ∗∗∗∗p < 0.0001. C, representative γ-H2AX foci are shown in control (vector) and RAD52 shRNA-expressing U2OS cells treated or untreated with 50 μM PDS for 48 h (left). γ-H2AX foci/nucleus in PDS-treated and -untreated cells were qualified and plotted (middle). The p value is indicated as ∗∗∗∗p < 0.0001. RAD52 Western blot was performed to show RAD52 depletion by shRNA with KU70 as a loading control (right). D, U2OS cells expressing vector or shRNA for RAD52 (left) and U2OS and U2OS-derived RAD52 KO cells (right) were treated with or without PDS (50 μM, 24 h), followed by γ-H2AX Western blot analysis using GAPDH as the loading control. DAPI, 4′,6-diamidino-2-phenylindole; DSB, double-stranded break; EGFP, enhanced GFP; G4, G-quadruplexes; PDS, pyridostatin.
Fig 4: XPF recruitment to G4 is dependent on RAD52.A, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-RAD52 before and after PDS (50 μM, 48 h) or CX-5461 (1 μM, 48 h) treatment. Enrichment of RAD52 at G4 was calculated using the ChIP value in PDS-untreated cells as 1 for normalization. B, U2OS [HR-EGFP (TPG4)] cells expressing Flag-RAD52 were depleted for MUS81 by shRNAs with vector (Vec) as the control. Anti-Flag ChIP analysis at G4 locus was performed before and after CX-5461 (1 μM, 48 h) treatment. Enrichment of XPF at G4 locus was calculated using the ChIP value in CX-5461–untreated cells with vector control as 1 for normalization (left). MUS81 depletion is shown by Western blot with KU70 as a loading control (right). C, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-XPF before and after PDS (50 μM, 48 h) or CX-5461 (1 μM, 48 h) treatment. Enrichment of XPF at G4 was calculated using the ChIP value in untreated cells as 1 for normalization. D, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-XPF and with or without depletion of RAD52 by shRNAs upon PDS (50 μM, 48 h) treatment. Enrichment of XPF at G4 was calculated using the ChIP value in the vector control as 1 for normalization (left). RAD52 depletion is shown by Western blot with KU70 as a loading control (right). In all experiments, error bars represent the SD of at least three independent experiments. ChIP, chromatin immunoprecipitation; EGFP, enhanced GFP; G4, G-quadruplexes; HR, homologous recombination; PDS, pyridostatin.
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