Topoisomerases relax DNA supercoils during replication, maintaining DNA integrity. Deficiencies cause DNA breaks, leading to neurological diseases and ageing. Camptothecin, a Top1 inhibitor, traps Top1cc at the DNA 3’end, preventing re-ligation and causing double-strand breaks when encountering replication forks. CPT, topotecan, and irinotecan are potent chemotherapeutic agents. Reduced Top1 levels in mammals are thought to protect cells from CPT.
In this study, I used Danio rerio, a vertebrate model organism, to examine the roles of nuclear Top1 paralogues (top1a, top1b) and the mitochondrial paralogue (top1mt) in a whole-animal setting. CRISPR/Cas9-mediated genome editing was used to generate top1a and top1b null mutant lines via microinjection, followed by the establishment of a top1a;top1b double mutants. Phenotypic, biochemical, and transcriptomic analyses (including RNA-seq and qPCR) were performed under basal conditions and following CPT treatment.
I found that loss of top1a produced viable larvae with developmental abnormalities and increased sensitivity to CPT. This was observed with reduced TOP1cc accumulation, challenging the assumption that CPT toxicity is directly linked to TOP1cc levels. Upregulation of top1b in top1a mutants suggested partial compensation, though it was insufficient to fully restore function. Conversely, top1b mutants exhibited no obvious phenotype or increased CPT sensitivity, suggesting a role. In double mutants, the phenotype matched that of top1a mutants, and they remained sensitive to CPT, confirming that top1a is the primary nuclear paralogue.
Transcriptomic profiling showed minimal changes under basal conditions, indicating developmental buffering. CPT induced transcriptional reprogramming, dominated by p53- and apoptosis induction, with limited activation of DNA repair pathways in top1-deficient backgrounds. Notably, top1mt was selectively induced during severe top1 loss and stress, but functional assays indicated limited compensation.
These findings show that zebrafish tolerate nuclear Top1 loss through partial paralogue compensation but primarily respond to genotoxic stress with apoptosis rather than repair, suggesting an alternative, Top1-independent cytotoxic mechanism.
