Fluctuations of alternative end-joining throughout the cell cycle with emphasis in Mitosis

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are repaired mainly by classical non-homologous end-joining (c-NHEJ) and homologous recombination (HR). When these pathways are compromised, cells may use another form of endjoining that operates as a backup, the so-called alternative end-joining (alt-EJ) repair pathway. In addition, single strand annealing (SSA) may also engage under certain conditions and for certain regions in the genome. While c-NHEJ is active throughout the cell cycle, HR functions only in Sand G2-phase of the cell cycle. Alt-EJ and SSA may function throughout the cell cycle, but they are more active in G2-phase. The efficiency of alt-EJ during early/mid S-phase remains unclear. Notably, several reports in literature suggest that all forms of DSB processing are suppressed during M-Phase. These reports are in contrast to earlier studies from our laboratory, but also to studies of other investigators, showing efficient repair of DSBs in M-phase cells. The current study aims to address two key questions: How does the efficiency of alt-EJ fluctuate from early/mid S-phase up to G2-phase of the cell cycle, and what is the actual ability of mammalian M-phase cells to repair DSBs and which repair pathways are involved? Pulse field gel electrophoresis (PFGE) experiments were carried οn highly synchronized A549 cells in early S-phase. Cells were exposed to 20Gy of X-rays and DSB repair kinetics were analyzed up to 4h post irradiation. In order to improve the accuracy of PFGE analysis, a new mathematical analysis method was developed, which takes into consideration the sensitivity changes of PFGE, as cells replicate their DNA. Using this method of PFGE analysis in synchronized early S-phase cells and upon suppression of c-NHEJ, the amount of DSBs was found to be reduced by almost 80% 4h after irradiation, similar to G2 synchronized cells. Hence, upon suppression of c-NHEJ, cells have the ability to repair DSBs with the same efficiency during Sand G2-phase ...