Abstract 4724: Regulation of DNA cleavage by the amino and carboxyl domains of human Top2α

DNA topoisomerases are involved in processing DNA structures such as knots and supercoils formed during cellular processes. hTop2α functions by creating transient DNA double-strand breaks, through which a second DNA molecule can be passed. DNA breakage by hTop2α involves formation of a transient covalent DNA-protein intermediate that can be stabilized by small molecules like etoposide and doxorubicin. Therefore, these agents convert Top2 into a DNA damaging agent. The overall architecture of type II topoisomerases has been defined by a combination of biochemical and structural studies. The homodimeric eukaryotic Top2 includes an N-terminal domain that includes an ATP dependent dimerization and ATPase activity, a domain that connects the ATPase domain to the catalytic core, termed the transducer domain, a central breakage/reunion core that carries out metal ion dependent formation of the protein/DNA covalent complex and a C-terminal domain that includes a second dimerization domain. Since drug resistance to Top2 targeting drugs can occur by mutations that greatly reduce enzyme activity, they are of limited usefulness in understanding detailed mechanisms of drug action. We carried out a large-scale screen to identify etoposide hypersensitive mutations in hTop2α. We developed novel approaches to target small regions of the hTop2α coding sequence. As anticipated, we identified several domains of hTop2α that were hotspots for changes leading to etoposide hypersensitivity. These regions included the N-terminal region of the protein (transducer and ATPase domain), the breakage/reunion catalytic core of the enzyme, and C-terminal dimerization domains. For example, we identified a prominent class of mutations including Asp374Gly, Asp374Glu, Asn445Asp, Val415Leu and Thr377Ile in the transducer domain, which confer hypersensitivity to etoposide. We purified and characterized the Asp374Gly mutant protein. In addition to an increased in vitro sensitivity to etoposide, Top2α (Asp374Gly) had a decreased requirement for ATP compared to the wild-type enzyme. The decreased requirement for ATP is unexpected and may suggest an alteration in the communication between the ATPase domain and the catalytic core. In addition, we also isolated etoposide hypersensitive alleles close to the C-terminal dimerization domain. These mutations included Asp1130Asn, Ala1052Gly and Gln1109Lys. Our results suggest at least two distinct controlling modules for Top2 cleavage: the ATPase/transducer module, and the C-terminal dimerization domain. Our results provide additional insight into how etoposide inhibits Top2α and may suggest new domains of the protein suitable for drug targeting. Citation Format: Abhishek Dilip Deshpande, Matthew A. Gilbertson, Hannah N. Miles, Karin C. Nitiss, John L. Nitiss. Regulation of DNA cleavage by the amino and carboxyl domains of human Top2α [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 4724.