Development Of Novel Visual-plus Quantitative Analysis Systems For Studying DNA Double Strand Break Repairs In Zebrafish

One of the most toxic insults in DNA is the double stranded breaks (DSB). Improper repair of such damage results in genomic instability which predisposes an organism to immunodeficiency, neurological damage and cancer. As such, organisms have evolved three efficient systems to repair such damage. They are Homologous Recombination (HR), Non-Homologous End Joining(NHEJ) and Single Strand Annealing (SSA) pathway.The use of reporter systems to analyze DNA double-strand break repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Scel, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for HR, SSA and NHEJ at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Scel recognition sites in opposite orientations rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding l-Scel-cut constructs, and the repair of DNA lesion caused by I-Scel could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB break sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore, while these HR and SSA reporter systems could be effectively decreased by the knockdown of rad51and rad52respectively, NHEJ could only be impaired by the knockdown of ligaselV (lig4) when the NHEJ construct was cut by I-Scel in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrated that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.