Volume 28, Issue 3, Pages 468-481 (November 2007) RAD51AP1 Is a Structure-Specific DNA Binding Protein that Stimulates Joint Molecule Formation during RAD51-Mediated Homologous Recombination  Mauro Modesti, Magda Budzowska, Céline Baldeyron, Jeroen A.A. Demmers, Rodolfo Ghirlando, Roland Kanaar  Molecular Cell  Volume 28, Issue 3, Pages 468-481 (November 2007) DOI: 10.1016/j.molcel.2007.08.025 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 DNA Damage Sensitivity Profile of HeLa Cells after siRNA-Mediated Downregulation of RAD51AP1 (A–D) Survival curves in response to increasing doses of (A) mitomycin C, (B) cis-platin, (C) ionizing radiation, and (D) camptothecin. siRNA#1 and siRNA#2 are targeted against RAD51AP1, whereas siRNA-L is a control siRNA targeted against luciferase. Note that the percentage of surviving cells is plotted on a logarithmic scale. (E) Survival curves in response to increasing doses of mitomycin C after RAD51AP1, RAD51, and their combined downregulation. (F) Survival curves in response to increasing doses of mitomycin C after RAD51AP1, XRCC3, and their combined downregulation. (G) Survival curves in response to increasing doses of ionizing radiation after RAD51AP1, RAD51, and their combined downregulation. (H) Survival curves in response to increasing doses of ionizing radiation after RAD51AP1, XRCC3, and their combined downregulation. (I and J) Relevant immunoblots detecting RAD51AP1 and the loading control GRB2. (I) and (J) show immunoblots from cell populations used in (B) and (D), and (A) and (C), respectively. (K) Relevant immunoblots detecting RAD51AP1, RAD51, and XRCC3 and the loading control GRB2 in cell populations used in (E)–(H). All cell survival experiments were performed in triplicate, and errors bars represent standard errors of the mean. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Spontaneous and Damage-Induced RAD51 Foci Formation Does Not Depend on RAD51AP1 (A) Colocalization of GFP-RAD51AP1 (green) and RAD51 (red, indirect immunofluorescence) into spontaneous and DNA damage-induced nuclear foci (3 hr after irradiation with 20 Gy). At least 95% of RAD51AP1 foci colocalized with RAD51 foci in both untreated and ionizing radiation treated cells. (B) Immunoblot analysis of extracts from HeLa cells downregulated for RAD51AP1 and collected just prior to treatment with DNA damaging agents in the experiment presented in (C) and (D). Detection of GRB2 was used as loading control. siRNA#1 and siRNA#2 are targeted against RAD51AP1, and siRNA-L is a nonspecific control siRNA against luciferase. (C) RAD51 foci formation as detected by immunofluorescence was quantified by counting the fraction of cells containing ≥5 foci/nucleus. UT, untreated; IR, ionizing radiation-treated; and MMC, mitomycin C-treated cells. (D) Representative micrographs obtained by superimposing the RAD51 signal (red) onto the DAPI DNA counter stain (blue). (E) Kinetics of RAD51 foci formation after RAD51AP1 downregulation was determined by counting the fraction of cells containing ≥5 foci/nucleus at the indicated time points after treatment with ionizing radiation (6 Gy). Immunoblot analysis to verify RAD51AP1 downregulation as in (B), of one of the experiments from which the data was collected, is shown as an inset. The average of two experiments is reported where error bars represent standard deviations. At least 100 cells were scored for each experimental point. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 RAD51AP1 Is an Elongated Monodisperse Monomer in Solution (A) Reducing SDS-PAGE gel of fractions after S Sepharose chromatography stained with Coomassie. Ni-NTA pool, fraction obtained by batch purification over Ni-NTA resin; FT, flowthrough; W, wash; and MW, molecular weight standard. (B) Coomassie-stained reducing SDS-PAGE gel of fractions after Sephacryl 200 gel permeation chromatography. (C) A semilog plot of the partition coefficient (Kav) versus molecular weight was constructed using a standard set of globular proteins to determine the apparent molecular weight of RAD51AP1. The HOP2/MND1 heterodimer with an expected mass of 50,180 Da was used as control. (D) Sedimentation equilibrium profiles at 4.0°C plotted as a distribution of the absorbance at 280 nm versus r at equilibrium. Data were collected at 10 (red), 13 (green), 16 (pink), and 19 (blue) krpm at a loading A280 of 0.90. The solid lines show the best-fit global analysis in terms of a single ideal solute, with the corresponding residuals shown in the panels above the plot. (E) Mass spectrometry of RAD51AP1 after electrospray ionization of the “native” protein in 50 mM ammonium acetate. Averaged calculated mass = 39,417 Da and experimental deconvoluted mass = 39,286 ± 6 Da, which corresponds exactly to the mass of the polypeptide minus the first methionine. The peaks marked with an asterisk represent a higher mass species and are absent when the protein is sprayed from a denaturing methanol solution, indicating a noncovalently bound adduct to the protein (data not shown). (F) Coomassie-stained reducing SDS-PAGE gel of RAD51AP1 after BS3 crosslinking. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 RAD51AP1 Specifically Stimulates D Loop Formation by RAD51 (A) Effect of RAD51AP1 in RAD51-catalyzed D loop assays with homologous invading oligonucleotide SK3 (lanes 1–12) or with nonhomologous invading oligonucleotides NHA (lanes 13–15) or NHB (lanes 16–18). Position of the radiolabel on the substrate is indicated with an asterisk. Recipient substrate was supercoiled pUC19 plasmid (SC pUC19). (B) Effect of RAD51AP1 in RAD51 (left) or DMC1 (right) catalyzed D loop assays with homologous oligonucleotide SK3. (C) Effect of RAD51AP1 (left) or C-terminally deleted RAD51AP1Δ327–352 (right) in RAD51-catalyzed D loop assays with homologous invading oligonucleotide SK3. (D) RAD51AP1 specifically and directly binds RAD51, but not DMC1. Immunoblot analysis (IB) of RAD51AP1-RAD51 binding reactions after immunoprecipitation (IP) as indicated (upper two panels). Immunoblot analysis (IB) of RAD51AP1-DMC1 binding reactions after immunoprecipitation (IP) as indicated (lower two panels). Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 RAD51AP1 Exhibits Structure-Specific DNA Binding (A) Electrophoretic mobility shift assays performed with, from top to bottom, ss, ds, splayed-arm, and Holliday junction DNA substrates at 2.5 nM. DNA substrates were constructed with the indicated oligonucleotides described in Table S2. Position of the radiolabel on the substrate is indicated with an asterisk. (B) Discrete retarded complexes contained both the radiolabeled DNA substrate (left panel, radioactivity prior to immunoblotting) and RAD51AP1 (right panel, anti-RAD51AP1). The DNA substrate used was the Holliday junction described above. (C) High-affinity DNA binding of RAD51AP1 depends on the presence of a branch in the DNA substrate rather than on the DNA sequence. Electrophoretic mobility shift assays performed with eight splayed-arm DNA structures, with different arm sequences as indicated in red or blue and ss and ds oligonucleotides as controls (upper panel). The concentration of the DNA substrate was 2.5 nM. The concentration of RAD51AP1 was at 1000 nM and titrated down in 2-fold increments. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 High-Affinity Binding of RAD51AP1 to Single-End Invasion DNA Substrates (A) Electrophoretic mobility shift assays performed with, from left to right, linear ds, bubble, D loop, ss branch, or ds branch containing D loop DNA substrates used at 2.5 nM final concentration. Position of the radiolabel on the substrate is indicated with an asterisk. (B) Fold RAD51AP1-mediated stimulation measured in RAD51-catalyzed D loop assays performed with an unbranched (SK3NT), ss (SK3), or ds (SK3+SK3-c) branch containing invading oligonucleotides. Averages of four experiments are plotted. Error bars represent standard deviations. (C) Dissection of SEI DNA intermediates into their respective left and right branched sites (top). Summary of the relative affinity of RAD51AP1 for various branched-DNA substrates as measured by electrophoretic mobility shift assays (bottom). Primary data is shown in Figure S4. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 RAD51AP1 Deletion Mutant Analysis (A) Scheme of the RAD51AP1 deletion variants produced for this analysis. (B) A reducing SDS-PAGE gel stained with Coomassie containing wild-type RAD51AP1 and deletion variants Δ327–352, Δ1–242, and Δ1–283. (C) RAD51 binding analysis of the RAD51AP1 deletions by in vitro coimmunoprecipitation as revealed by immunoblots. Anti-RAD51AP1 (R1085) was reactive against all deletion variants, including Δ327–352 (data not shown). Its effectiveness was reduced for deletion variants Δ1–242 and Δ1–283, possibly due to loss of available epitopes. (D) Effect of RAD51AP1 deletion variants in RAD51-catalyzed D loop assays using homologous invading oligonucleotide SK3 and supercoiled pUC19 plasmid as recipient. Quantification is reported in Table S1. (E) Analysis of the RAD51AP1 deletion variants by mobility shift assays using 5 nM of radiolabeled SEI DNA substrate with a ds protruding arm. RAD51AP1 was titrated starting at 2000 nM and diluted in 2-fold increments. Quantification is reported in Table S1. Molecular Cell 2007 28, 468-481DOI: (10.1016/j.molcel.2007.08.025) Copyright © 2007 Elsevier Inc. Terms and Conditions