Analysis of Topoisomerase-induced DNA Breaks in Zebrafish

Topoisomerase and oxidative DNA breaks (TODs) disrupt transcription, causing neurological disease and age-associated degeneration. TODs can be caused by the aberrant activity of topoisomerases, which transiently cleave the DNA to resolve supercoiling. The proximity of the reversible topoisomerase-DNA complexes to oxidative breaks converts them to toxic and irreversible DNA breaks. The enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase-linked DNA breaks and a variety of oxidative DNA breaks. The protein mutated in ataxia telangiectasia, ATM, has also been implicated in the repair of TODs. The progressive accumulation of TODs leads to pathology, most notably progressive degeneration of specific tissues in the nervous system. To study this process at the whole organismal level, we have used CRISPR/Cas9 to generate zebrafish models lacking Tdp1 and Atm. The tdp1^-/- zebrafish are viable, do not have increased DNA damage at embryonic stages and show mild neurological phenotypes in adulthood. Like mice and human cells, the adult tdp1^-/- zebrafish are hypersensitive to the topoisomerase 1 (Top1) poison topotecan. However, surprisingly, the embryos of tdp1^-/- zebrafish are not more sensitive to topoisomerase poisons than their wild-type siblings. This suggests that the adult fish rely on Tdp1 to tolerate elevated levels of Top-breaks whereas alternative compensatory mechanism/s are more efficient in embryos. Elucidating the compensatory pathways is important for our understanding of selective vulnerability of certain neuronal populations to TODs and will shed light on our understanding of organismal ageing.