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

Purpose : To measure rejoining of radiation-induced double-stranded DNA-fra gments 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 wi th photons or accelerated nitrogen ions (linear energy transfer, LET = 125 keV mum(-1)) and incubated for repair for 0-22 h. Double-stranded DNA-fragm ents from the irradiated cells were separated by pulsed-field gel electroph oresis in the range similar to5 kbp to 5.7 Mbp. Results: For cells irradiated with high LET nitrogen ions, there was an inc rease in the fast half-time from similar to5 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 th reshold sizes. For cells irradiated with lower doses of low LET radiation t he 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 do es not detect small fragments will result in an underestimation, or even di sappearance of the fast-rejoining phase. This is due to substantial rejoini ng of fragments taking place before the fragments are of sufficient size to be monitored.