Spatiotemporal dynamics of the DNA double strand break marker 53BP1 after exposure to ionising radiation

Abstract

53BP1 is known to be involved in the DNA damage response and has been shown to localise into discrete foci at the site of DNA double strand breaks (DSBs) after exposure to ionising radiation (IR). Quantification of radiation induced foci (RIF) at varying times after exposure has been used to assess the induction of DSB and kinetics of their decline, suggestive of repair. In addition, the size and relative nuclear distribution of foci, observed at different times after IR, could provide insights into the dynamics of these foci that may be relevant for understanding mechanisms of chromosome exchange. To assess this human bronchial epithelial (HBEp) cells, which are the primary target for 50% of our average annual radiation exposure, have been irradiated with low linear energy transfer (LET) 60Co γ-rays (0- 2.0 Gy) or 238Pu high LET α-particles (~1α-particle/nucleus LET 121-235 keV/μm). DSB were quantified at various time points (0-24hrs) after IR, stained for 53BP1 and categorised into three sizes (<0.5μm, 0.5 μm-1.0 μm and >1.0μm in diameter).The data generated was used to ask questions on the radiobiological DSB response of HBEp cells (chapter 4), the recruitment of 53BP1 to DSB and the possible dynamics of DSB for repair. In addition, immortalised HBEp cells transfected with 53BP1-GFP fusion protein were irradiated with 60Co γ-rays and analysed to further assess the spatio-temporal aspects of DSB in live cells. After exposure to 2Gy γ-rays peak induction of 53BP1 was observed within 30 min (22foci) with a subsequent decline to sham levels (2 foci) after 24hrs. For the quantification of spatiotemporal dynamics (chapter 5), a bespoke foci analysis tool was developed (chapter 3) to provide detailed measurements of RIF number, size and relative location with greater speed and reliability than manual counting and categorising method. This novel approach to foci analysis provides evidence for limited (<1μm) movement of foci is presented that may support the ICN model for chromosomal exchange aberrations.