Exonuclease 3

Chromosome-orientation fluorescence in situ hybridization (CO-FISH) was employed to evaluate IR-induced chromosomal instability and performed as previously described (62 (link), 70 (link)) with some modification. Following irradiation, cell cultures were incubated for various times, trypsinized, and subcultured into medium containing 5-bromo-2-deoxyuridine (BrdU, 10 μM; Sigma-Aldrich) for one cell cycle. Slides were stained with Hoechst 33258 (0.50 ng/μL; Sigma-Aldrich) for 15 min and exposed to 365 nm UV light (Stratalinker 2400) for 25 min. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min. Slides were hybridized with a Cy-3 conjugated (TTAGGG)₃ PNA telomere probe (0.2 μg/mL; Applied Biosystems) at 37°C for 1.5 h, rinsed in 70% formamide at 32°C for 10 min, and dehydrated in another ethanol series before re-probing at 37°C for 2 h. Following the second hybridization, slides were rinsed with 70% formamide at 32°C for 15 min followed by 5 min rinse in PN buffer. Chromosomes were counterstained with DAPI (4,6-diamidine-2-phenylindole dihydrochloride; Vectashield, Vector Laboratories). Preparations were examined and images captured and analyzed using a Zeiss Axioskop2 Plus microscope equipped with a Photometrics Coolsnap ES2 camera and running Metavue 7.1 software.

Cells were cultured in a medium containing a 3:1 ratio of 5′-bromo-2′-deoxyuridine (BrdU, Sigma):5′-bromo-2′-deoxycytidine (BrdC, Sigma) at a total final concentration of 10 μM during the final 24 h. Colcemid addition led to the accumulation of mitotic cells. Cultures were trypsinized and then treated with hypotonic KCl, fixed, and dropped onto microscope slides. Prior to hybridization of the single-stranded telomere probe (as above for FISH), slides were treated with 0.5 mg/ml RNase A (Sigma) for 10 min at 37 °C and then stained with 0.5 μg/ml Hoechst 33258 (Sigma) in 2× SSC for 15 min at room temperature. Slides were then exposed to 365 nm UV light for 25 min. The BrdU/BrdC-substituted DNA strands were digested with 3 U/μl of exonuclease III (Promega) in a buffer supplied by the manufacturer (50 mM Tris-HCl, 5 mM MgCl₂, and 5 mM dithiothreitol, pH 8.0) for 10 min at room temperature. An additional denaturation was performed in 70% formamide, 2× SSC at 70 °C for 1 min, followed by dehydration in a cold ethanol series (70, 85, and 100%). The CO-FISH procedure results in the original parental strands serving as single-stranded chromosomal target DNA for hybridization of single-stranded probes.

Cells were treated with 0.5 μg/ml of Colcemid (Invitrogen) for 4 h before harvest. Chromosomes were fixed with 3% formamide and hybridized with a telomere PNA-FISH 5′-Cy3-OO-(CCCTAA)₃–3′ probe (PNAgene) as described in Wu et al. (10 (link)). DNA was counterstained with DAPI. Digital images were captured using NIS-Elements BR (Nikon) with a Nikon Eclipse 80i microscope utilizing an Andor CCD camera. The relative telomere signals were analyzed with ImageJ software (downloaded from Fiji). For CO-FISH, cells were incubated with 10 μM BrdU for 12 h, treated with 0.5 μg/ml of Colcemid for 4 h and harvested. Formalin-fixed metaphase spreads were stained with 0.5 μg/ml of Hoechst 33258 (Sigma) in 2 × SSC for 15 min at room temperature before being exposed to UV light equivalent to 5.4 × 10³ J/m². After digestion with 200 U of Exonuclease III (Promega), the samples were denatured at 85°C for 3 min and incubated sequentially with 5′-Cy3-OO-(CCCTAA)₃–3′ and 5′-FAM-OO-(TTAGGG)₃–3′ probes as described above. Images were captured as described above.