Investigation of switch from ATM to ATR signaling at the sites of DNA damage induced by low and high LET radiation

•ATM and ATR are oppositely regulated by length of single stranded overhangs generated during end processing by nucleases.•High LET radiation-induced DNA damage leads to sustained resection at break sites.•Sustained resection leads to extended transition from ATM to ATR (concomitant NHEJ to HR) at sites of DNA damage.•Prolonged transition from ATM to ATR signaling/sustained resection is due to complex damage induced by high LET radiation.•This prolonged transition may contribute to increased biological effectiveness of high LET radiation. Upon induction of DNA damage by ionizing radiation (IR), members of the phosphatidylinositol 3-kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM), DNA-PKcs, and ATM- and Rad3-related (ATR) maintain genomic integrity by mounting DNA damage response (DDR). Recent reports suggest that activation of ATM and ATR are oppositely regulated by the length of single stranded overhangs generated during end processing by nucleases at the break sites. These stretches of single stranded overhangs hold the clue for the transition from ATM to ATR signaling at broken DNA ends. We investigated whether differential processing of breaks induced by low and high LET radiation augments the phenomenon of switching from ATM to ATR kinase and hence a concomitant NHEJ to HR transition at the sites of DNA damage. 82-6 human fibroblasts were irradiated with 1 or 2Gy of γ-rays and particle radiation of increasing LET in order to increase the complexity and variability of DNA double strand breaks (DSB) structures. The activation kinetics of ATM and ATR kinases along with their downstream substrates were determined utilizing Western blotting and immunofluorescence techniques. Our data provide evidence of a potential switch from ATM to ATR kinase signaling in cells treated with γ-rays at approximately 2h post irradiation, with induction and completion of resection denoted by Rad51 foci resolution kinetics and observed with a significant decline of phosphorylated ATR kinase 8h after IR. On the other hand, irradiation with high LET 600MeV/u 56Fe (180keV/μm) and 170MeV/u 28Si (99keV/μm) particles show a similar Rad51 foci decay kinetics, however, exhibiting prolonged resection, evident by the persistent phosphorylated ATM and ATR kinase until 24h post irradiation. This residual effect, however, was significantly reduced for 250MeV/u 16O particles of moderate LET (25keV/μm) and absent for γ-rays. Hence, our results support the hy