Topoisomerase II (Top2) is an essential enzyme that binds DNA to produce a transient double strand break (DSB) that allows the release of topological stress (supercoiling) and catenations that arise mostly during DNA mitotic replication. Since Top2 binds covalently to DNA, the amount of DNA-bound Top2 must be tightly controlled as these complexes impede the progression of DNA polymerases, causing replication fork collapse, unscheduled DSB formation and genome instability. In fact, Top2 poisons that prevent Top2 removal from DNA, such as etoposide, are key components of anticancer drugs. However, the mechanisms that regulate the amount of DNA-bound Top2 are not understood. We show that in C. elegans and human cells lacking aminopeptidase P (APP-1/ (XPNPEP1 in humans), which removes the N-terminal amino acid of peptides carrying a proline in position 2, display an increase in cellular levels of Top2, demonstrating that APP-1 is a key regulator of Top2 levels. Importantly, this overall increase in Top2 levels causes accumulation of Top2 on DNA in humans and worms, suggesting that Top2 activity is modulated through the control of its cellular levels. app-1 mutant worms display reduced fertility, genomic instability, and a dramatic accumulation of RAD-51 recombination intermediates in their germlines. RAD-51 intermediates persist even in the absence of SPO-11, the topoisomerase-like enzyme that induces DSBs during meiosis to promote inter-homologue crossover events, demonstrating that APP-1 prevents the formation of unscheduled DSBs in the germline. Knockdown of XPNPEP1 in human cells also causes formation of unscheduled DSBs. Analysis of transgenic strains demonstrates that the enzymatic activity of APP-1 is required for genome stability and to prevent TOP-2 accumulation, while analysis of DNA repair in app-1 mutants reveals the presence of replication-related damage, suggesting that excess TOP-2 impairs the progression of DNA polymerases. In fact, app-1 mutants are hypersensitive to etoposide, but resistant to top-2 knockdown by RNAi, confirming that they accumulate functional TOP-2. Thus, APP-1 is a conserved regulator of Top2 levels and increased amounts of Top2 cause genome instability. These findings could have therapeutic implications, since increasing the cellular levels of Top2 should sensitize cells to Top2 poisons commonly used to treat a variety of human cancers.
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