Low dose ionizing radiation strongly stimulates insertional mutagenesis in a gamma H2AX dependent manner

Abstract

Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of the stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (gamma H2AX) and is not reduced by DNA polymerase theta (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies gamma H2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations, and warrants further exploration in vivo to determine if the phenomenon has implications for radiation risk assessment.Author summaryNot all DNA in mammalian nuclei is organized into chromosomes. The pool of extrachromosomal DNA molecules is produced by natural process: during genomic DNA repair, viral infections, phagocytosis; and in experimental settings after transfection, and in gene therapy. Extrachromosomal DNA can integrate into the genome at the site of a double-stranded DNA break (DSB), producing a mutation in the chromosome. Because DSBs are dangerous lesions, they are actively eliminated, and their availability is a limiting factor for extrachromosomal DNA integration. It has long been known that inducing additional random DSBs, for example by exposing cells to ionizing radiation, can increase the frequency of random integration (RI), however the irradiation doses that were used were non-physiological. We found that much smaller doses of radiation, in the upper range of radiological diagnostic procedures, stimulate integration even more efficiently than the much larger doses studied previously. The potency of stimulation is remarkable, given that biological effects of such low doses are generally difficult to register, and warrants re-evaluation using animal models. Surprisingly, the genetic dependencies of radiation-stimulated integration do not include DSB repair pathways, and are distinct from background integration events. Our observations provide a hyper-sensitive tool to detect mutagenesis and reveal new information about the genetic interactions between DNA damage signaling and repair system components.