Differential Recruitment of DNA Repair Proteins KU70/80 and RAD51 upon Microbeam Irradiation with alpha-Particles

Simple Summary: Human populations can be exposed to ionizing radiation (IR) in different circumstances (natural, medical, and industrial). By interacting with the matter, IR causes damage in all cellular compartments, including DNA. Mammalian cells possess several different DNA repair mechanisms for removing IR-induced DNA damage among which DNA double-strand breaks (DSBs) are the most deleterious lesions. In this work, we investigated the temporal dynamics of the two major DSB repair pathways upon alpha-particle irradiation delivered by the MIRCOM microbeam. We found that these mechanisms are differentially recruited at IR-induced DNA damage sites. In addition to representing a significant part of the natural background radiation exposure, alpha-particles are thought to be a powerful tool for targeted radiotherapy treatments. Understanding the molecular mechanisms of recognition, signaling, and repair of alpha-particle-induced DNA damage is not only important in assessing the risk associated with human exposure, but can also potentially help in identifying ways of improving the efficacy of radiation treatment. alpha-particles (He2+ ions), as well as other types of ionizing radiation, and can cause a wide variety of DNA lesions, including DNA double-strand breaks (DSBs). In mammalian cells, DNA DSBs can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, we investigated their dynamics in mouse NIH-3T3 cells through the recruitment of key proteins, such as the KU heterodimer for NHEJ and RAD51 for HR upon localized alpha-particle irradiation. To deliver alpha-particles, we used the MIRCOM microbeam, which allows targeting of subnuclear structures with submicron accuracy. Using mouse NIH-3T3 cells, we found that the KU heterodimer is recruited much earlier at DNA damage sites marked by H2AX phosphorylation than RAD51. We also observed that the difference in the response of the KU complex and RAD51 is not only in terms of time, but also in function of the chromatin nature. The use of a microbeam such as MIRCOM, represents a powerful tool to study more precisely the cellular response to ionizing irradiation in a spatiotemporal fashion at the molecular level.