Fig 1: The FA-Derived RFWD3 I639K Mutant Cannot Interact with RPA(A) U2OS cells stably expressing GFP-RFWD3 (WT or a catalytically inactive C315A mutant) or GFP only were exposed to HU (2 mM) or MMC (50 ng/mL) for 16 hr and then lysed. Anti-GFP immunoprecipitates were probed with the indicated antibodies. In the case of IR, cells were exposed to 10 Gy and left to recover for 1 hr before lysis.(B) U2OS cells stably expressing GFP-RFWD3 (C315A or C315A I639K) or GFP only were exposed to the indicated concentrations of MMC for 16 hr and then lysed and analyzed as in (A).(C) The human RFWD3 amino acid sequence was entered into the WD40 repeat prediction algorithm at the WDSPdb database. The table predicts six WD40 repeats (WD 1–6). For each repeat, the sequence of the four anti-parallel ß strands typical of WD40 repeats is given, as well as the sequence of loops connecting these ß strands. In red are shown predicted surface hotspots that, in other WD40 domain proteins, mediate key protein-protein interactions.(D) Same as (B), except that U2OS cells expressing GFP-RFWD3 C315A or GFP-RFWD3 C315A bearing mutations in the predicted WD40 hotspots described in (C) were treated with a single dose of MMC for 16 hr before analysis.
Fig 2: Development of non-inhibitory UbV binders for human E3 ligases. (A) Schematic representation of the UbV nomination process. Within the UbV library, each phage particle displays a unique UbV. Binding phages were captured with immobilized E3 ligases. After a total of five rounds of phage display selection, individual binding clones were subjected to sequencing. UbV binders were then characterized for their effects on known E3 ligase biological functions, and a total of six non-inhibitory UbVs were selected for UbVIP construction. (B) Protein sequences of the non-inhibitory UbVs (UbVRF.5, UbVRF.7, UbVRF.9, UbV3A.2, UbV3A.3, UbV3A.5, UbVNL.1, and UbVMDM2). Only the substitutions across the randomization surface of wild type Ub (WT Ub) are shown. It should be noted that UbVs have two amino acid extensions at the C-terminal (position 77 and 78). Dashes indicate conservation of the WT Ub sequence. (C) Binding curves of RFWD3 UbVs and RPA2 to RFWD3WD40, measured by ELISA (n = 3). The half maximal binding concentrations (EC50) of UbVs and RPA2 to RFWD3WD40 were determined by established methods [15] and are listed in the text. RFWD3WD40 (1 µM) were immobilized in microtiter plates. Serial dilutions of FLAG-tagged UbV or GST-tagged RPA2 (0–4 µM) were added and incubated for 20 min at room temperature. Wells were washed, and bound UbV/RPA2 was detected by anti-FLAG/GST-HRP conjugate antibody and colorimetric development of TMB peroxidase substrate. The absorbance at 450 nm (y-axis) was plotted against Log (UbV/RPA2 concentration, nM) (x-axis). (D) RPA2 binding to RFWD3WD40 was not affected by UbVRF.5, UbVRF.7, or UbVRF.9. Competition ELISA assays were performed as described in (C) for binding between RFWD3WD40 (immobilized and pre-incubated with UbVs or RPA2. (E) Binding curves of UBE3A UbVs to UBE3AHECT, measured by ELISA (n = 3). Experiments were conducted as described in (C). (F) UBE3AHECT protein (pre-mixed for 15 min with WT Ub or UbV as indicated) was incubated for 1 h at room temperature with E1 (UBE1), E2 (UBE2L3), ATP, and Ub. Western blots were probed with an anti-Ub antibody (clone FK2) to detect mono- and poly-ubiquitinated UBE3AHECT. UbVs are not incorporated into chains because their C termini do not contain a di-glycine motif that is required for recognition by the E1 enzyme. (G) Binding curves of UbVMDM2 to MDM2RING, measured by ELISA (n = 3). Experiments were conducted as described in (C). (H) As in (F), autoubiquitination of MDM2RING was assessed with control (WT Ub) or UbVMDM2.
Fig 3: RFWD3 Interaction-Defective RPA Mutants Attenuate ICL Repair(A) Alignment of the C-terminal region of RPA32 orthologs from the indicated species. Conserved residues are highlighted in red. Residues tested by mutational analysis are denoted by asterisks.(B) Anti-FLAG precipitates from extracts of U2OS cells stably expressing FLAG-RPA32 WT or FLAG-RPA32 bearing the indicated mutations were subjected to western blotting with the indicated antibodies.(C) U2OS cells stably expressing FLAG-RPA32 WT or FLAG-RPA32 bearing the indicated mutations were transfected with an siRNA targeting the 3′ UTR of the RPA32 gene. These cells were then subjected to the indicated doses of MMC, and a clonogenic survival assay was carried out. For each cell type, the viability of untreated cells is defined as 100%. Data are represented as mean ± SEM; n = 3.(D) U2OS cells stably expressing FLAG epitope only (EMPTY), FLAG-RPA32 WT, or FLAG-RPA32 bearing the indicated mutations were transfected with an siRNA targeting the 3′ UTR of the RPA32 gene. Cells were then left untreated (mock) or treated with MMC (2ng/mL) for 48 hr before ethanol fixation, propidium iodide staining, and FACS analysis.
Fig 4: RFWD3-Deficient Cells Show Hallmarks of Defective ICL Repair(A) Clonogenic survival analysis of control and RFWD3?/? HeLa cells exposed to the indicated genotoxins. For each cell type, the viability of untreated cells is defined as 100%. Data are represented as mean ± SEM; n = 3. Control cells are parental cells that were taken through genome editing protocols but are WT for RFWD3. Circles, control; squares, RFWD3?/?.(B) Cells were left untreated (mock) or treated with MMC for 24 hr before ethanol fixation, propidium iodide staining, and fluorescence-activated cell sorting (FACS) analysis. The proportion of cells in G2 phase is indicated in each case.(C) Metaphase spreads were prepared from cells of the indicated genotypes that were left untreated or treated with MMC for 24 hr. The number of chromosome breaks and radial chromosomes per metaphase spread was quantified. Data are represented as mean ± SEM.
Fig 5: Two UbVIPs recruited cellular E3 ligases to induce 53BP1 degradation. (A) Experimental outline for determining 53BP1 protein expression in mammalian cells. HEK 293 cells were transfected with the UbVIP constructs and cells lysed after 24 h and whole cell lysates were subjected to SDS-PAGE and immunoblotting using an anti-53BP1 antibody. (B) Western blot analysis of 53BP1 expressed in HEK 293 cells compared to untransfected control, presence of UbVIP was confirmed by blotting for FLAG. ß-Actin was used as a loading control. The UbVIP-i53#3 and #5 corresponding to UbVRF.9-8aa-i53 and UbVNL.1-8aa-i53 respectively, caused a reduction in 53BP1 levels when compared to UbVIP with the same targets but different orientation. 53BP1 protein abundance was measured using ImageJ software and normalized to ß-Actin. Values indicated are relative to the untransfected control. (C,D) UbVIP-i53#3 and #5 interact with 53BP1 and corresponding E3 ligases (RFWD3 and NEDD4L). Immunoprecipitation (IP) of Flag-tagged proteins from extracts prepared from HeLa cells transfected with vectors expressing UbVIP-i53 #3 and UbVIP-i53 #5 or the empty vector control. Proteins were separated by SDS-PAGE and immunoblotted (IB) for FLAG, 53BP1, RFWD3 in (C) and NEDD4L in (D).
Supplier Page from Abcam for Anti-RFWD3 antibody