Phosphoimager

RNA was extracted from cell line pellets and human tumor biopsy samples using TRIreagent (Sigma-Aldrich, Munich, Germany) or TRIzol (Life Technologies). Samples were DNase treated, further phenol-chloroform extracted and ethanol precipitated. Samples (typically 3 µg) were mixed (1:1) with Ambion gel loading solution and separated by 15% dPAGE in MOPS buffer and 7M urea. Ambion “decade” ssRNA size markers were used (Thermo Fisher Scientific, Grand Island, NY, USA). Gels were ethidium bromide stained, blotted (by semidry electroblotting) and the RNA cross-linked to the membranes using EDC (1-ethyl-3-dimethylaminopropyl carbodimide) at 60 °C for 90 min as described [45 (link)]. Blots were prehybridized in 2x SSC, 7% SDS, 200 µg/mL ssDNA at 50 °C. RNA complementary oligomer probes were radiolabeled using the mirVana kit (Life technologies), or full length EBER1 using T7 transcribed from EBER1 template with ³²P-UTP. Hybridization was at 50 °C overnight. Blots were washed in 0.1 × SSC, 0.1% SDS at 50 °C and visualized using a phosphoimager GE Healthcare Life Sciences, Freiburg, Germany).

DNA in Ku or MRE11 complexes was measured as previously described (24 (link)). Briefly, 10⁷ cells were treated with 1 Gy of IR or ⁵⁶Fe, ³⁷Si. Protein–DNA complexes in treated cells were cross-linked with 1% formaldehyde. Cells were resuspended with buffer A (50 mM Tris, pH 8.1, 1% SDS; and 10 mM EDTA), followed by sonication. The samples were immunoprecipitated using Ku70 or Mre11 antibody, respectively in buffer B (16.7 mM Tris,pH 8.1, 1% SDS; 1% Triton X-100; 1.2 mM EDTA; 167 mM NaCl). The immunoprecipitation complexes were washed one time with buffer C (20 mM Tris, pH8.1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA and 150 mM NaCl), buffer D (500 mM NaCl) and buffer E (10 mM Tris, pH 8.1, 25% LiCl, 1% NP40, 1% deoxycholic acid and 1 mM EDTA), then two times with Tris–EDTA buffer (pH 8.0). DNA in Ku70–DNA or Mre11–DNA complexes was labeled with [γ-³²P]ATP by incubation with T4 polynucleotide kinase in kinase buffer at 37°C for 45 min and then at 65°C for 10 min. The samples were then washed four times with TE buffer (pH 8.0) and incubated in TE buffer containing 1 mg/ml protease K (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 2 h. The samples were separated on a 5% non-denaturing polyacrylamide gel. DNA signals were detected and analyzed using a PhosphoImager with ImageQuant software (GE health, Waukesha, WI).

2D gel analysis of replication intermediates was carried out as described previously 59 (link). DNA was prepared and digested in agarose plugs with the indicated restriction enzymes 12 (link). Enriched fractions for replication intermediates were obtained using BND cellulose columns (SIGMA). Gels were blotted onto Hybond-N+ nylon membrane. The probes were labeled with alpha-³²P dCTP and the blot analyzed on a PhosphoImager and quantified with the ImageQuant software (pause/ arc + pause in percentage) (GE Healthcare Life Sciences). Sequences of primers used to produce the probe are indicated in Table 2.

Psoralen cross-linking^{12 (link)} was performed in isolated nuclei that were irradiated in the presence of 10 μg/ml 4,5,8’-trimethylpsoralen (Sigma–Aldrich). Genomic DNA was isolated and 10 μg was digested with SalI (Promega). Fragmented DNA was separated on a 0.9% agarose gel, transferred onto nylon membranes, and immobilized by UV irradiation at 1875 x 100 μJ/cm2 using a UV cross-linker (Stratalinker 2400; Agilent Technologies). The membrane was hybridized with ³²P-labelled rDNA (generated using a nick translation labeling kit, Roche) and visualized on a PhosphoImager (GE Healthcare). Quantification was performed using ImageQuant (TLv2005.04; GE Healthcare). Original psoralen blot scans that correspond to quantitated and/or edited blot images shown in Figs 1, 2 and Supplementary Fig. 2 are shown in Supplementary Fig. 1a, b and Supplementary Fig. 8. A representative depiction of the psoralen blot quantitation is shown in Supplementary Fig. 1c, d.