Rejoining of double-stranded DNA-fragments studied in different size-intervals

PURPOSE: To measure rejoining of radiation-induced doublestranded DNA-fragments of different sizes and to evaluate the effects of size-resolution in the analysis of rejoining. MATERIAL AND METHODS: Normal human fibroblasts (GM5758) were irradiated with photons or accelerated nitrogen ions (linear energy transfer, LET = 125 keV microm(-1)) and incubated for repair for 0-22 h. Double-stranded DNA-fragments from the irradiated cells were separated by pulsed-field gel electrophoresis in the range approximately 5 kbp to 5.7 Mbp. RESULTS: For cells irradiated with high LET nitrogen ions, there was an increase in the fast half-time from approximately 5 min for fragments < 400 kbp to 10 min when all fragments < 5.7 Mbp were measured. Further, the fraction of fragments rejoined by the slow-rejoining phase increased significantly for increased threshold sizes. The fraction of unrejoined fragments after 22 h and the half-time for the slow-rejoining phase remained constant for all threshold sizes. For cells irradiated with lower doses of low LET radiation the rejoining was shifted towards a slower kinetics when fragments up to 10 Mbp were excluded in the analysis. CONCLUSION: DNA exclusion-size and resolution may affect the estimates of DNA double-strand break rejoining. Using a low-resolution technique that does not detect small fragments will result in an underestimation, or even disappearance of the fast-rejoining phase. This is due to substantial rejoining of fragments taking place before the fragments are of sufficient size to be monitored.