Abstract 1742: Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases

Abstract

Abstract Type II topoisomerases play key roles in transcription, replication and chromosome segregation. These enzymes modulate DNA structure by generating transient DNA breaks where the protein is covalently bound to DNA. Anti-cancer drugs targeting topoisomerase II (Top2) inhibit the religation of enzyme-induced breaks, and exert their anti-cancer effects through the generation of enzyme mediated DNA damage. In addition to small molecule inhibitors, it has been suggested that aberrant DNA structures or DNA lesions may also lead to Top2/DNA lesions. A mammalian Top2 isoform, Top2β has been suggested to play a key role in hormone-mediated transcription, and in carrying out this function, generates potentially oncogenic DNA damage. We have assessed the generation of potentially oncogenic DNA damage by Top2β in model organisms, and using the purified protein. We found that ectopic expression of Top2β is poorly tolerated in repair deficient yeast strains, while expression of the mammalian Top2α isoform lacks this property. We measured the induction of homologous recombination by Top2β in yeast and found that ectopic expression of the enzyme led to elevated levels of recombination. Recently, a de novo mutation in Top2β was reported in a patient with severe developmental delay resulting from an His63Tyr substitution in Top2β 1. We found that Top2β (His63Tyr) could not complement a defect in yeast Top2, but encoded an enzyme that was catalytically active, and induced elevated levels of recombination. Because of the inferred location of His63 in the ATPase domain of Top2β, and proximity to the ATP binding site of the enzyme, we hypothesized that the mutant protein was partially defective in ATP regulated DNA cleavage. To further explore how ATP regulates Top2β cleavage, we examined the effects of expressing a Top2β variant completely lacking the N-terminal ATPase domain of Top2β. While this mutant did not complement yeast top2 mutants, we found that it s expression induced 10-20 fold higher levels of recombination than cells expressing wild type Top2β. This result indicates that an ATP deficient variant of Top2β induces very high levels of DNA damage. We suggest that regulation of the ATPase activity of Top2β may be critical for maintaining genome stability, and may be responsible for induction of DNA damage during hormone-mediated transcription. 1. C.W. Lam et al., Global developmental delay and intellectual disability associated with a de novo TOP2B mutation. Clinica Chimica Acta 469:63-68, 2017. Citation Format: Amanda M. Johnson, Lokha Ranjani A. Boopathy, Raveena Gupta, Hannah N. Miles, Matthew Gilbertson, Karin C. Nitiss, John L. Nitiss. Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1742.