Enhanced DNA end resection and subsequent responses during the processing of complex DNA double strand breaks induced by heavy ion beams.

There are two major DNA double strand break (DSB) repair pathways in human cells, non-homologous end-joining (NHEJ) and homologous recombination (HR). Although the actual mechanisms regulating repair pathway choice are still largely unknown, it has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. It is known that high linear energy transfer (LET) radiations, such as heavy ion beams, induce complex DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished.An early step in HR is the generation of 3’-single strand DNA (ssDNA) via a process called DNA end resection. A critical player in this process is CtIP, which is phosphorylated by ATM and initiates resection together with Mre11 nuclease. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed ssDNA. After heavy ion beam irradiation, massive phosphorylation of CtIP and RPA2 arises. More than 80 % of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20-40 % of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends drastically alters the balance towards resection-mediated repair. Since it has been well accepted that the ssDNA exposed by resection can be a signal for the activation of ATR kinase, we further investigated how the enhanced resection influences the function of ATR pathway. Our results of G2/M checkpoint assays show that ATR pathway plays a pivotal role and functions in a dose- and LET-dependent manner to regulate the early G2/M arrest in ATM-deficient cells. Collectively, our study reveals that the complexity of DSB end structure has s drastic influence on repair pathway choice, and provokes the cellular signaling regulated by ATR. In addition, we found novel characteristics of CtIP at DSB foci following the initiation of resection, suggesting that CtIP has functions in addition to promoting the initiation of resection during HR. We will discuss an overview of the cellular responses to heavy ion beam irradiation.ICRR2015 (15th International Congress of Radiation Research