TDP1 drives MRE11-dependent and independent end resection during replication stress by TOP2 poisons

DNA-Topoisomerase crosslinks are lesions formed by proteins covalently bound to DNA, and these complexes directly impede transcription and replication, thereby threatening genomic integrity. Tyrosyl-DNA phosphodiesterase 1 (TDP1) and MRE11 remove DNA-protein adducts, but their functional relationship remains unclear. We show TDP1 and MRE11 act epistatically in the removal of etoposide-stimulated topoisomerase II cleavage complexes (TOP2cc) in S/G2 phases of the cell cycle. Etoposide-stimulated TOP2cc formed ahead of and behind replication forks, inhibiting fork progression. Consistently, replication fork progression was decreased in cells lacking TDP1 or upon inhibition of MRE11 nuclease activity, and a similar defect was observed when combining both conditions. Furthermore, TDP1 promotes DNA end resection downstream of MRE11 and facilitates nascent strand degradation at stalled forks independently of MRE11 nuclease activity. Accordingly, combined TDP1 loss and MRE11 inhibition did not exacerbate etoposide hypersensitivity. We provide evidence that TDP1 stimulates replication-coupled removal of TOP2cc by facilitating DNA end processing through both MRE11-dependent and -independent pathways. Overall, our results suggest TDP1 promotes nucleolytic excision of TOP2-mediated replication blocks, in addition to its canonical hydrolase activity.