Cell Culture, Reagents, and Antibodies
HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), and 2% glutamine. U2OS/DR-GFP cells were grown in McCoy's media supplemented with 10% FBS. The antibodies used were anti-RPA2 (Calbiochem), anti-phospho-RPA2 (S4, S8) (Bethyl Laboratories), anti-phospho-RPA2 (S33) (Bethyl Laboratories), anti-ATR (Santa Cruz), anti-ATM (Santa Cruz), anti-DNA-PKcs (Thermo Scientific), anti-CHK1 (Santa Cruz), anti-CHK2 (Santa Cruz), anti-phospho-CHK1 (Ser 345) (Cell signaling), anti-phospho-CHK2 (Thr 68) (Cell signaling), anti-γH2AX (Upstate), anti-H2AX (Abcam), anti-HA (HA-7, Sigma). The SMART pools of all gene-specific siRNAs were purchased from Dharmacon. MMS, HU, 4NQO, camptothecin were purchased from Sigma.
Construction of Various Expression Plasmids
Both wild type RPA2 and the S4A S8A mutant RPA2 used in this study contain an HA epitope tag. HA-RPA2 S4A, S8A was generated by site-directed mutagenesis (Stratagene) with two primers GAC CAA GAT GTG GAA CGC TGG ATT CGA AGC CTA TGG CAG CTC CTC and GAG GAG CTG CCA TAG GCT TCG CCA GCG TTC CAC ATC TTG GTC.
Detection of Chromatin-bound RPA2
HEK293T, MO59K, MO59J, HCT116 (a gift from Dr. Eric Hendrickson's laboratory, University of Minnesota), Seckel, or AT cells (purchased from Coriell Institute) (as shown in and respectively) were treated with DNA damaging agents for four hours before the cells were harvested as indicated in figures. When cells were treated with either IR or UV, cells recovered for four hours after irradiation and were then harvested. To make the chromatin-bound fraction, approximately 107 cultured cells were resuspended in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M Sucrose, 10% Glycerol, 1 mM PMSF, 5 µg/mL aprotinin, 20 µg/mL leupeptin) and Triton X-100 was added (to a final concentration of 0.1%). Precipitated chromatin-bound fractions obtained by centrifugation at 4°C at 1,300×g for 5 min were then resuspended in TSE500 buffer [20 mM Tris (pH 8.1), 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100, protease inhibitor cocktail (Roche)] and sonicated. Chromatin-bound proteins were collected from the supernatant after centrifugation at 17,000×g for 5 min.
RAD51 Immunofluorescence Microscopy
105 HEK293T cells plated on two-well chamber slides were transfected with siRNA specifically targeting the 3′-UTR of RPA2 (Dharmacon, SMART pool, #9, #11, #12), together with a plasmid expressing either siRNA-resistant HA-tagged RPA2 or RPA2 S4A, S8A and cultured for three days. The cells were then irradiated with 5 Gy of γ-irradiation followed by a 6 hr recovery period and then fixed with 3.5% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100 for 10 min. Fixed cells were blocked with 5% FBS and stained with anti-RAD51 and rhodamine-conjugated anti-mouse secondary antibodies.
Flow Cytometry Analyses
Two days post transfection (to allow cells to replace the endogenous RPA2 in HEK 293T cells as described above), cells were incubated with 2 mM of HU for 22 hr. Cells were then cultured in fresh medium without HU but with 0.5 µg/ml of nocodazole for 12 hr. BrdU was added at 1 hr before harvest. Cells were then fixed and stained with both APC-conjugated anti-BrdU and FITC-conjugated anti-phospho-H3 antibodies. Cells were sorted by Becton Dickinson FACSCaliburs as described 
siRNA and Recombination Reporter Assay
U2OS human osteosarcoma cell lines stably transfected with a single copy of an intact DR-GFP reporter gene were used to measure the HR frequency 
. The endogenous RPA2 was replaced with HA-tagged RPA2 or RPA2 S4A, S8A as described above. Two days later, a plasmid pCAG-I-SceI (which expresses I-SceI enzyme), or an empty vector, pCAG, together with a pDsRed monomer (as a transfection efficiency control) were transfected. HR frequency was determined by the number of cells expressing GFP divided by the number of cells expressing DsRed monomer. Experiments were repeated at least three times and the average values are reported.
Pulsed field Gel Electrophoresis
~2.5×106 of HEK293T cells were treated with DNA damaging agents for 4 hr. Cells were then trypsinized and melted into SeaPlaque GTG agarose (Cambrex Bio Science Rockland, Inc) with 0.75% agarose final concentration. Agarose plugs were then digested in proteinase K reaction buffer (100 mM EDTA, pH 8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, and 1 mg/ml Proteinase K) at 50°C for 2 days and washed 4 times in wash buffer (20 mM Tris, pH 8.0, 50 mM EDTA). The plugs were loaded onto a 1% pulsed field certified agarose (Biorad). Separation was performed on a CHEF-DR III pulsed field electrophoresis systems (Biorad; 120 field angle, 240 s switch time, 4 V/cm, 14°C) for 24 hr. Gels were stained with ethidium bromide and DSBs were quantified and analyzed using fluorescence image analyzing system FLA 5100 (FujiFilm).
Cell Survival Assay
Cell viability was determined in HEK293T cells where endogenous RPA2 was replaced by WT-RPA2 or RPA2 S4A, S8A. Approximately 6000 cells were plated in triplicate on 96-well plates. 4NQO was added into the medium16 hr after plating. Cell viability was determined three days later by the CyQUANT Cell proliferation assay kit (Invitrogen).