假設依據：
Ku and DNA-PKcs are required for joining of both RS and coding ends during V(D)J recombination . Because our results showed that c-Myc suppresses both Ku and DNA-PKcs activities , it is also possible that c-Myc may negatively affect V(D)J recombination.
Measurement of Ku70/86 Activity
Ku70/Ku86 DNA binding activity was analyzed using a Ku70/86 DNA Repair Kit (Active Motif). Briefly, cells were washed and resuspended in hypotonic buffer. The nuclear extract was isolated for Ku activity analysis. Five micrograms of nuclear protein was loaded into the oligonucleotide-coated 96-well plate and incubated for 60 minutes at room temperature. Ku70 or Ku86 antibody was added and incubated for another 60 minutes. After washing, HRP-conjugated secondary antibody was added and incubated for 60 minutes. After washing, developing solution and stop solution were added. Optical density was read on a spectrophotometer at 450 nm. Each experiment was repeated three times and data represent the mean ± SD of three separate determinations.
Pulsed-Field Gel Electrophoresis
Pulsed-field gel electrophoresis (PFGE) was performed as described previously [29]. Briefly, cells were harvested, resuspended in ice-cold buffer L [0.1 MNa2-EDTA, 0.01 MTris, 0.02MNaCl (pH 8.0)] at a concentration of 5 × 106 cells/ml, and mixed with an equal volume of 1% low melting point agarose (Beckman) at 42°C. The mixture was pipetted into a small length of Tygon tubing, clamped tight at both ends, and chilled to 0°C. The solidified agarose 「snake」 was extruded from the tubing, added to 10× volume of buffer L containing 1 mg/ml proteinase K and 1% sarkosyl, and incubated for 16 hours at 50°C. Following lysis, the agarose snake was washed four times with TE buffer and then cut into 0.5-cm plugs. The plugs were inserted into the wells of a pre-cooled 1% low melting point agarose gel (4°C). PFGE (200-s pulse time, 150 V, 15 hours at 14°C) was performed using Clamped Homogenous Electric Fields Mapper (Bio-Rad, Hercules, CA). After electrophoresis, the gel was stained with ethidium bromide for photography.
Telomere–Fluorescence In Situ Hybridization
Analysis Telomere–Fluorescence In Situ Hybridization (T-FISH) was performed using Telomere peptide nucleic acid (PNA) FISH Kit/Cy3 (DakoCytomation, Copenhagen, Denmark) as described [30]. Cells were incubated with colcemid (KaryoMAX; Gibco, Grand Island, NY) at 100 ng/ml for 1.5 hours and then harvested by trypsinization. Cells were swollen in pre-warmed 30 mM sodium citrate for 30 minutes at 37°C, fixed in methanol/acetic acid (3:1), and air dried on slides overnight.
After pepsin digestion, slides were denatured at 80°C for 5 minutes, hybridized with Cy3-labeled PNA telomeric probe (Cy3-[TTAGGG]3) in 70% formamide at room temperature for 3 to 4 hours, washed, dehydrated, and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Metaphase images were captured using a fluorescent microscope (Zeiss) and ×63 objective lens. At least 30 metaphases of each cell line were scored for chromosomal aberrations.
Transient V(D)J Recombination
Assay Recombination substrates pJH200 and pJH290 were used in the transient V(D)J recombination assay to test for RS and coding joins as described [31]. Briefly, pJH200 or pJH290 was co-transfected into cells with the full-length RAG1 and RAG2 expression constructs under the control of the Rous sarcoma virus long terminal repeat promoter using Lipofectamine 2000. Extrachromosomal DNA was recovered after 48 hours by alkaline lysis as previously described [32] and electroporated into E. coli MC1061. The V(D)J recombination activity was measured by comparing the ratio of ampicillinresistant (Ampr) and chloramphenicol-resistant (Camr) colonies versus Ampr colonies in cells. Because a precise signal join generates an ApaLI site, ApaLI digestion of polymerase chain reaction products from the recombinant substrate pJH 200 was used to measure the fidelity of the RS joins as described [31]. The coding join sequence was determined by comparison of the nucleotide sequence isolated from individual recombined pJH290 clones.