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97 UHRF1 siRNA: 5′-GUCAUUUACCACGUGAAAUUU-3′
Antibodies and dilution factors
The dilutions of the various antibodies for Western blot analysis were 1:1000 for anti-PWWP2B (Abcam, ab110052), 1:1000 for anti-UHRF1 (Santa Cruz, SC-373750), 1:1000 for anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA: F3165), 1:2000 for anti-Myc (Roche, Basel, Switzerland;
11814150001), 1:5000 for anti-GFP (Clontech 632380), 1:2000 for anti-β-actin (Sigma-Aldrich, A5316), 1:2000 for anti--tubulin (Millipore, 05829), 1:1000 for anti-RAD51 (Abcam, ab63801), 1:1000 for anti-RPA2 (Bethyl 22 3 1 Laboratories, Inc., A300-244A), 1:1000 for anti-RPA2 (S4/8) (Bethyl Laboratories, Inc., A300-244A), 1:1000 for anti-BRCA1 (Santa Cruz, SC-6954), 1:1000 for anti-53BP1 (Cell Signaling Technology, #4937), and 1:1000 for anti-RIF1 (Bethyl Laboratories, Inc., A300569A). Horseradish peroxidase-conjugated secondary antibodies specific to rabbit (Sigma- Aldrich, A0545) or mouse (Sigma-Aldrich, A9917) IgG were used at a dilution of 1:2000. The dilutions of the various antibodies for indirect immunofluorescence analysis were 1:200 for anti- PWWP2B (Novus Biologicals, NBP1-55176), 1:500 γH2AX (Millipore, 05-636), 1:200 for anti- UHRF1 (Santa Cruz, SC-373750), 1:200 for anti-RAD51 (Abcam, ab63801), 1:200 for anti-BRCA1 (Santa Cruz, SC-6954), 1:200 for anti-53BP1 (Cell Signaling Technology, #4937), 1:1000 for anti- RPA2 (Bethyl Laboratories, Inc., A300244A), 1:100 for anti-RPA2 (S4/8) (Bethyl Laboratories, Inc., A300-244A), 1:1000 for antiMRE11 (Novus Biologicals, NB100-142) and 1:100 for anti-Myc (Roche, 11814150001) (Roche).
Transfection and immunoprecipitation
Transient transfection was performed using poly (ethylenimine). For immunoprecipitation, the cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed in NETN buffer (0.5%
Nonidet P-40, 20 mM Tris [pH 8.0], 50 mM NaCl, 50 mM NaF, 100 µM Na3VO4, 1 mM dithiothreitol (DTT), and 50 µg/ml phenylmethylsulfonyl fluoride (PMSF)) at 4 °C for 10 min. The crude lysates were cleared by centrifugation at 14,000 rpm at 4 °C for 5 min, and the supernatants were incubated with protein A-agarose-conjugated primary antibodies. The immunocomplexes were washed three times with NETN buffer and subjected to sodium dodecyl sulfate4 1 polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed using the antibodies indicated in each figure legend.
98 Cell survival assay
HeLa cells in a 60 mm-diameter plate were transfected twice with the indicated siRNAs at 24-h intervals. Forty-eight hours after the second transfection, the transfected cells were irradiated at the indicated doses using a radiation source. Fourteen days after irradiation, the cells were washed with PBS, fixed, stained with 2% (w/v) methylene blue, and the colonies were counted.
GST pull-down
Glutathione S-transferase (GST) fusion protein or GST was expressed in Escherichia coli and immobilized on glutathione-Sepharose 4B beads. The cell lysates of the HEK293T cells transfected with plasmids encoding the indicated proteins were incubated with the GST fusion protein or GST for 1 h at 4°C. After washing with NETN buffer, the samples were subjected to SDS–PAGE. Western blotting was performed using the antibodies indicated in each figure legend.
Laser microirradiation and imaging of cells
The accumulation of GFP-UHRF1 wild-type or mutants, and GFP-PWWP2B wild-type or mutants was analyzed as previously described 21.
Homologous recombination and non-homologous end-joining assay
The homologous recombination assay was performed as previously described 22. U2OS DR-GFP or EJ5-GFP cells were first treated with the indicated siRNAs in 24-well plates and then transfected with 0.4 µg of I-SceI per well 24 h after siRNA transfection. Seventy-two hours after I-SceI transfection, the cells were trypsinized, and the percentage of GFP-positive cells was determined by flow cytometry. Statistical analysis was performed by the paired t-test. BD FACSCanto II (NFEC-2011-02- 145106) is used for FACS analysis at the Core Facility Center for Chronic and Metabolic Diseases at Sookmyung Women’s University.
Quantitative analysis of end resection using ER-AsiSI U2OS cells
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Quantitative analysis of end resection using ER-AsiSI U2OS cells was performed as previously described 23. ER-AsiSI U2OS cells were grown in Dulbecco’s modified Eagle’s medium containing 10%
FBS. siRNA transfection of ER-AsiSI U2OS cells was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s instructions. The ER-AsiSI U2OS cells were treated with mock or 4-hydroxytamoxifen (4-OHT, Sigma; H7904) for 4 h. The ER-AsiSI U2OS cells were collected at a concentration of 1.2 × 107 cells/ml with 37°C 0.6% low-gelling point agarose (Bio-Rad) in PBS. A 50 µl cell suspension was dropped on Parafilm (Pechiney) to make a solidified agar ball, then transferred to a tube. The agar ball was treated with 1 ml of ESP buffer (0.5 M EDTA, 2% N- lauroylsarcosine, 1 mg/ml proteinase-K, and 1 mM CaCl2, pH 8.0) for 20 h at 16°C with rotation, followed by treatment with 1 ml of HS buffer (1.85 M NaCl, 0.15 M KCl, 5 mM MgCl2, 2 mM EDTA, 4 mM Tris, and 0.5% Triton X-100, pH 7.5) for 20 h at 16°C with rotation. After washing with 1 ml of PBS for 6 h at 4°C with rotation, the agar ball was melted by placing the tube in a 68°C heat block for 10 min. The melted sample was diluted 15-fold with 68°C ddH2O and mixed with an equal volume of the appropriate restriction enzyme buffer. The resection near the specific DSBs was measured by quantitative polymerase chain reaction (qPCR) analysis. The genomic DNA sample (36 µl) was digested with 80 units of BsrG1 or HindIII (New England Biolabs) restriction enzymes at 37°C overnight. The digested samples (3 µl) were used as templates in a 25-µl qPCR containing 12.5 µl of TaqMan Universal PCR Master Mix (ABI), 0.5 mM of each primer, and 0.2 mM probe using a QuantStudio 7 Flex Real-Time PCR System (ABI). The percentage of ssDNA generated by resection at the selected sites was calculated as previously described (Zhou et al, 2014). For each sample, the Ct was calculated by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample. The percentage of ssDNA was calculated by the following equation: ssDNA (%) = 1/(2(4Ct−1) + 0.5) * 100.
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