Enhancement of Repeat-Mediated Deletion Rearrangement Induced by Particle Irradiation in a RecA-Dependent Manner in Escherichia coli

SIMPLE SUMMARY: Repeat-mediated deletion (RMD) is generally induced by a DNA double-strand break (DSB), but the effect of DSB complexity on RMD initiation is unclear. In order to address this issue, this research firstly used an Escherichia coli reporter line in which amp restoration was controlled by lacI repeats. Then, particle irradiation was used to stochastically generate complex DSBs. The results clearly confirmed the enhancement of RMD rearrangement induced by proton and carbon irradiation in a dose- and LET-dependent manner. Meanwhile, RMD rearrangement was suppressed by intermolecular homology, in which the length of homology is more important than the composition of homology. Exogenous recombinase could significantly promote particle irradiation-induced RMD events. The RecA-dependent pathway was suggested to be involved in the enhancement of RMD under particle irradiation. These results could broaden our understanding of RMD generation and help assess the repair process of complex DSBs located around the repeat sequences. ABSTRACT: Repeat-mediated deletion (RMD) rearrangement is a major source of genome instability and can be deleterious to the organism, whereby the intervening sequence between two repeats is deleted along with one of the repeats. RMD rearrangement is likely induced by DNA double-strand breaks (DSBs); however, it is unclear how the complexity of DSBs influences RMD rearrangement. Here, a transgenic Escherichia coli strain K12 MG1655 with a lacI repeat-controlled amp activation was used while taking advantage of particle irradiation, such as proton and carbon irradiation, to generate different complexities of DSBs. Our research confirmed the enhancement of RMD under proton and carbon irradiation and revealed a positive correlation between RMD enhancement and LET. In addition, RMD enhancement could be suppressed by an intermolecular homologous sequence, which was regulated by its composition and length. Meanwhile, RMD enhancement was significantly stimulated by exogenous λ-Red recombinase. Further results investigating its mechanisms showed that the enhancement of RMD, induced by particle irradiation, occurred in a RecA-dependent manner. Our finding has a significant impact on the understanding of RMD rearrangement and provides some clues for elucidating the repair process and possible outcomes of complex DNA damage.