IntroductionIonizing radiation refer to particles and electromagnetic waves (photons) whose energy is well above the electronvolt (eV) and thereby sufficient to remove tightly bound electrons from atoms, creating ions. They are widely encountered in medical imaging, cancer treatment, industrial applications, and natural or accidental environmental exposure. Their marked impact on living cells arises from their propensity to break covalent bounds. On chromosomal DNA, radiation-induced damage challenges the DNA repair mechanisms protecting the genome. More specifically, exposure to ionizing radiation can generate coincident DNA double-strand breaks (DSBs) within a single cell and thereby favours the resealing of DNA ends stemming from distinct breaks by the Nonhomologous End Joining repair pathway (NHEJ)1,2. Such inaccurate repair events result in chromosome rearrangements, including mitotically unstable dicentric and acentric chromosomes. This NHEJ-dependent mutagenesis stems from NHEJ’s limited ability to remain accurate when confronted to multiple DSBs simultaneously. It is the main driver of radiation-induced cell lethality and forms the basis for the use of ionizing radiation in cancer treatment.Along they pass through cells, ionizing radiation deposits their energy primarily through multiple ionization events. Each energy deposition event can damage DNA directly via ionization and indirectly by generating a transient, localized high concentration of reactive species—with timescales ranging from a few nanoseconds to a few microseconds and a diffusion range of a few nanometers3,4. DSBs arise from simultaneous attacks on both strands. The frequency of DSBs is proportional to the energy deposited, which corresponds the absorbed dose of ionizing radiation. The co-occurrence of DSBs within the same cell, and consequently the formation of NHEJ-dependent chromosome rearrangements, depends on the absorbed dose, the dose rate (radiation intensity), and the genome size. These processes are also influenced by the radiation quality, specifically the linear energy transfer of the ionizing particles traversing the cell nucleus.The timescale of standard irradiation is much longer than the half-life of the reactive species generated by ionizing radiation (minutes to seconds versus microseconds to picoseconds, respectively). In this context, energy deposition events initiated by distinct particle/photon-matter interactions are thought to be independent, as they occur sequentially. However, whether this independence holds when the radiation dose is delivered within a timescale comparable to the lifetime of the transient reactive species remains unknown. For instance, co-occurring reactive species from distinct tracks may cross-react, potentially altering their effects on organic molecules, including DNA5. This question is relevant to better understand how ionizing radiation induces DSBs and chromosome rearrangements, as well as for refining the molecular basis of high or ultra-high dose rate irradiation protocols currently being developed for cancer therapy, known as FLASH-radiotherapy3,6,7,8.To investigate the impact of extreme dose rate on radiation-induced DNA damage, we took advantage of a recently developed genetic assay designed to capture and quantify chromosome fusions in budding yeast (Fig. 1A). This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome rearrangements9. It is sensitive enough to quantify the basal rate of chromosome fusions occurring in unexposed wild-type cells. The CFC assay also captures NHEJ-dependent chromosome rearrangements induced by ionizing radiation.Fig. 1Impact of oxygen pressure on the frequency of radiation-induced chromosome rearrangements. (A) Schematic representation of the CFC assay9. Radiation-induced localized ionization and reactive species formation can lead to two closely spaced DSBs, resulting in inaccurate end synapsis and repair by NHEJ. This generates chromosome rearrangements, including dicentric and monocentric chromosomes. CEN6 elimination by the Cre recombinase stabilizes dicentric chromosomes formed by fusion between chromosome 6 and another chromosome. If chromosome 6 remains monocentric, CEN6 elimination produces an unstable acentric chromosome leading to cell death. (B) Representative images of colonies growing on selective plates following gamma-ray irradiation (137Cs) of stationary yeast cells under normoxic (21% O2) or hypoxic (