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 (γH2AX) and is not reduced by DNA polymerase θ (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 γH2AX 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.
Highlights
Extrachromosomal DNA–endogenous, viral or transfected–can integrate into the genomic DNA, resulting in an insertional mutation
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 doublestranded DNA break (DSB), producing a mutation in the chromosome
Summary
Extrachromosomal DNA (ecDNA)–endogenous, viral or transfected–can integrate into the genomic DNA, resulting in an insertional mutation. This type of mutagenesis has been primarily studied in the context of exogenous DNA that enters the nucleus as a result of transfection or viral infection, and has several important practical implications [1]. Background RI shown in supplementary and S-RI plots shown in the main figures represent all data collected in the low dose irradiation experiments. To produce the low dose S-RI plots, normalized data from repeat experiments was averaged and fitted with a sigmoid curve using the “[Agonist] vs response” function in GraphPad Prism software v8.
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