Dynamic Interplay of CNHEJ and MMEJ Pathways of DNA Double-strand Break Repair During Embryonic Development in Zebrafish

Double strand breaks (DSBs) are the most deleterious DNA lesions as they frequently result in mutations when repaired by canonical non homologous end-joining (cNHEJ) and microhomology-mediated end-joining (MMEJ). Here, we investigated the relative importance of cNHEJ and MMEJ pathways during zebrafish embryonic development. We have analyzed the expression of cNHEJ and MMEJ related genes and found that it was dynamic during development and often become increased in specific tissues. We showed that inactivation of nuclear DNA ligase 3 (nLig3) or DNA polymerase theta (Polθ), two key MMEJ factors, did not affect zebrafish development but sensitized embryos to ionizing radiations and that deficiency of Polθ, but not nLig3, profoundly alters the mutation spectrum induced during repair of Cas9-mediated DSBs. By contrast, inactivation of DNA ligase 4, required for cNHEJ, did not seem to sensitize embryos to ionizing radiations nor to affect repair of Cas9-mediated DSBs but resulted in important larval growth defects. Our study underscores the dynamic and context-dependent roles of cNHEJ and MMEJ pathways during zebrafish development, highlighting their differential requirements across developmental stages and in response to genotoxic stress.