Induction and rejoining of DNA double-strand breaks in human cells after exposure to ionising radiation: an experimental and modelling approach

The induction of DNA double-strand breaks (DSBs) by ionising radiation and subsequent rejoining were studied in normal human fibroblasts in vitro, using radiation of different qualities. Radiation of increasing linear energy transfer (LET) can induce local hot spots of energy deposition that may result in DNA damage that is difficult to repair, and can potentially be mis-repaired. Due to the spatial association of the energy deposition events after the passage of ionising radiation, double-strand breaks (DSBs) and other types of DNA damage may be induced at genomic distances that reflect chromatin structure and radiation quality, introducing higher orders of damage complexity. The pulsed field gel-electrophoresis (PFGE) technique was used to resolve the fragmentation patterns after induction and rejoining of DSBs, separating double–stranded DNA fragments between 30 kbp and 5·7 Mbp. Several analytical and numerical methods of quantification of DNA damage were used and critically analysed. A novel method of DSB quantification based on random breakage was developed, which was applied to an extensive data–set along with several other methods. Results of this comparative analysis suggest that conventional methods of DSB quantification may be misleading, due to the way they handle the unwanted background damage that is produced during manipulation of the samples. Significant deviations from the random breakage predictions were observed for 238Pu α-particle irradiation, using a numerical method based on DSB clustering to analyse experimental data. In order to have an indication of the relative biological relevance of DSBs induced by radiation of different LETs, DSB rejoining kinetics were measured in living cells, and a detailed fragmentation analysis was carried out. A simple rejoining kinetics numerical model was designed and developed, based on a semi-empirical approach. The kinetic model was applied to the analysis of many experiments from this study. The results of the computer simulations are in good agreement with the experimental data. Even if the initial fragmentation patterns were significantly different for X-rays vs α-particles, DSBs induced by both radiations seem to rejoin according to a first order kinetics with two decay components, that do not depend on the size of the fragments being rejoined. For the molecular–weight size distribution that was observed in this project, the results indicate that the complexity of individual DSBs, rather than proximity effects between distinct DSBs, determine the rejoining kinetics