Reporter cassettes for detection of NHEJ and HR. (a) Reporter cassette for detection of NHEJ. The cassette consists of the GFP gene under CMV promoter with an engineered intron from the rat Pem1 gene, interrupted by an adenoviral exon (Ad). The adenoviral exon is flanked by I-SceI recognition sites in inverted orientation for induction of DSBs. In this construct, the GFP gene is inactive; however, upon induction of a DSB and successful NHEJ, the construct becomes GFP⁺. The construct also contains the hybrid neomycin/kanamycin-resistance gene and bacterial origin of replication. The cassette is flanked by EcoRI sites to facilitate rescue of reporter cassettes from genomic DNA. SD, splice donor; SA, splice acceptor. Shaded rectangles indicate polyadenylation sites. (b) Reporter cassette for detection of HR. The cassette consists of two mutated copies of GFP-Pem1. In the first copy of GFP-Pem1, the first GFP exon contains a deletion of 22 nt and an insertion of two I-SceI recognition sites in inverted orientation. The 22-nt deletion ensures that GFP cannot be reconstituted by an NHEJ event. The second copy of GFP-Pem1 is lacking the ATG and the second exon of GFP. Upon induction of DSBs by I-SceI, gene conversion events reconstitute active GFP gene. (c) Incompatible DNA ends generated by digestion of two inverted I-SceI sites.

Overexpression of TRF2 proteins in HCA2–hTERT cells. (a) Schematic diagram of the full-length TRF2 and the deletion mutants TRF2^ΔBΔM, TRF2^ΔB, and TRF2^ΔM. The numbers correspond to amino acid positions. (b) Western blot analysis of TRF2 overexpression. HCA2–hTERT cells were transfected with full-length TRF2, TRF2^ΔBΔM, TRF2^ΔB, or TRF2^ΔM using Amaxa electroporation. The cells were harvested at the indicated time after transfection. In each lane, 50 μg of total protein was loaded and hybridized with anti-TRF2 antibodies (see Materials and Methods). Equal loading was verified by hybridization with anti-Actin antibodies. (c) Expression of I-SceI in HCA2–hTERT cells. Cells were transfected with I-SceI-expressing plasmid and harvested at the indicated time, and 50 μg of total protein was analyzed by Western blot with anti-HA antibodies. Equal loading was verified by hybridization with anti-actin antibodies.

Effect of TRF2 overexpression on NHEJ (a) and HR (b). HCA2–hTERT cells containing NHEJ or HR reporter cassettes were cotransfected with the plasmids encoding the full-length or truncated TRF2 (5 μg), I-SceI (5 μg), and pDsRed2-N1 (0.1 μg). I-SceI induces DSBs in the GFP-based reporter cassettes (Fig. 1), and successful repair by NHEJ for NHEJ reporter or HR for HR reporter results in the appearance of GFP⁺ cells. To quantify NHEJ or HR events, the cells were analyzed by flow cytometry 4 days after transfection. The DsRed was used to normalize transfection efficiency. The ratio of GFP⁺ to DsRed⁺ cells was used as a measure of NHEJ or HR. The experiments were repeated at least five times. Error bars indicate SD, and asterisks indicate the treatments significantly different from controls (P < 0.01, Student's t test).

Effect of TRF2 depletion on NHEJ and HR. (a) Depletion of TRF2 by siRNA. HCA2–hTERT cells containing NHEJ or HR reporter cassettes were transfected with 100 pmol of anti-TRF2 or control siRNAs (SI00742630 and 1022076; Qiagen, Valencia, CA). The siRNA transfection was repeated twice with 3-day intervals. The amount of TRF2 was analyzed by Western blot at the indicated time after the second siRNA transfection. Each lane contains 50 μg of whole-cell protein extract. Actin was used as a loading control. (b) NHEJ and HR efficiency in TRF2-depleted cells. Reporter cell lines were transfected with I-SceI plasmid and DsRed2-N1 on day 3 after the second siRNA transfection. The ratio of GFP⁺ to DsRed⁺ cells serves as a measure of NHEJ or HR efficiency. The experiments were repeated four times. Error bars indicate SD. (c) Western blot showing Rad51 overexpression. Cells were transfected with a plasmid encoding Rad51 cDNA under CMV promoter. (d) Cell cycle distribution of TRF2-depleted cells on day 1 after I-SceI transfection. Cell cycle distribution was determined by PI staining and FACS analysis.

Effect of TRF2 depletion on formation of Rad51 foci after γ-irradiation. (a) Representative Rad51 foci in cells transfected with control or TRF2 siRNA. Cells were exposed to 8 Gy of irradiation and fixed for immunohistochemical analysis of Rad51 at the indicated times. Green is immunofluorescent staining of Rad51; blue is DNA stained with DAPI. (b) Numbers of Rad51 foci per nucleus in control siRNA or TRF2 siRNA-treated cells. The experiments were repeated three times, and 180 nuclei were counted per each experimental point. Error bars indicate SD.