Abstract
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are known as major DNA repair mechanisms involved in elimination of DNA double strand breaks (DSBs) induced by ionizing radiation. While NHEJ operates efficiently throughout the cell cycle, HR is efficient only in the S and G2 phases of the cell cycle. Recently, the existence of several pathways in NHEJ was revealed, and it was found that there are the DNA-PKcs/Ku-dependent pathway and the pathway requires artemis. Since roles of these two repair mechanisms have not been elucidated yet, we examined participation of these pathways to the repair of DSB induced by an introduction of restriction enzymes creating different types of broken ends.
Normal human cells synchronized in G0 phase were introduced by restriction enzymes (Pvu II, 100 U) by electroporation, and DSBs were determined by immunofluorescence staining for anti-53BP1 antibody. We found the foci of 53BP1 in 90% of the nuclei at 1 hour after introducing restriction enzyme. Among those foci-positive cells, 60 % of them had plenty of foci that were uncountable, and 20 % had 3-20 foci, which were called Type III foci. The number of Type III foci increased with time after introducing restriction enzyme in accordance with the decrease of cells harboring multiple foci, indicating repair of DSB. To distinguish artemis dependent-repair cells were treated with ATM inhibitor KU55933(KU). Although the ratio of cells showing Type III foci was not different, the foci number per nuclei were more in cells treated with KU. Thus, it was indicated that blunt-end type DSBs require the activity of artemis.
