Heat reduced mobility of unrejoined DNA fragments in NHEJ deficient cells. (A) NHEJ proficient cells (M059K, V3+wt DNA-PKcs, CHO-K1, AA8 and Irs1SF (XRCC3 mutant)) or (B) NHEJ deficient cells (M059J (DNA-PKcs mutant), V3 (DNA-PKcs mutant), Irs20 (DNA-PKcs mutant), GM16147 (XRCC4 mutant) and Xrs5 (Ku80 mutant)) were irradiated and were allowed to repair the DNA for up to 24 h. Naked DNA was produced either by a warm or cold DNA extraction protocol and was separated by PFGE. The amount of damage detected at t = 0 h in cells lysed by the cold protocol was set to 100% (cont. on opposite page). The damage above this level detected by the warm DNA extraction protocol at 0 h consists of non-true DSBs due to the inclusion of artifactual DSBs at 50°C; these non-true DSBs were repaired within 1 h after irradiation. (C) 24 h after irradiation M059J (NHEJ deficient) cells were lysed with four different protocols: 1. Warm DNA extraction (50°C, 18 h); 2. Cold DNA extraction (0°C, 2 × 18 h); 3. Warm DNA extraction (50°C, 18 h) + 2 M Salt (0°C, 18 h); 4. Cold DNA extraction (0°C, 2 × 18 h) + 0.5 M EDTA (50°C, 18 h). Error bars represent the SD of at least three experiments. *Denotes significant difference with paired t-test (p < 0.05) between warm and cold extraction.