Abstract 4853: Regulation of proteolytic repair of Top2 covalent complexes

Abstract

Abstract DNA topoisomerases (topos) play an essential role in nuclear processes such as replication, transcription, and chromosome segregation. These enzymes regulate DNA topology by introducing transient breaks in DNA with formation of transient protein/DNA covalent complexes. These complexes can be trapped by aberrant DNA structures, and are also the basis for anti-cancer drugs that target topos. Since topos can be trapped on DNA, leading to strand breaks with protein adducts at the site of the breaks, cells require DNA repair pathways to process protein adducts. We have examined the repair of the type II topos (Top2) in yeast and mammalian cells. We developed a sensitive assay to detect ubiquitylation and other post-translational modifications (PTMs) of Top2 trapped on DNA in S. cerevisiae and have shown that the proteasome plays a key role in repair of Top2 damage induced by the anti-cancer drug etoposide. We demonstrated that sumoylation of Top2 is induced by etoposide, and that deletion of the SUMO ligase Siz2 prevents etoposide induced sumoylation. Deletion of either of the genes encoding the ubiquitin ligase Slx5/Slx8 led to decreased ubiquitylation of trapped Top2. The mammalian ortholog of Slx5/Slx8 is Rnf4, and it too plays a role in ubiquitylation and degradation of Top2 trapped by etoposide. A human cell line completely lacking Rnf4 is defective in ubiquitylation of Top2β, one of the two mammalian Top2 isoforms. Interestingly, repair of the Top2α isoform is unaffected in an Rnf4 knockout cell line. Proteolysis of trapped Top2 is also subject to negative regulation. The UBA domain protein UBAP2L, (also termed NICE4) is amplified in various cancers. Knockout of the UBAP2L increases proteolysis of trapped Top2. Levels of trapped Top2α and Top2β are decreased in the absence of UBAP2L, and the level of trapped Top2 returns to the wild type level when MG132 is added, indicating UBAP2L regulates the proteasome mediated degradation of Top2. UBAP2L physically interacts with Top2β as determined by co-immunoprecipitation, and the interaction is not seen in Rnf4 knockout cells. Finally, knockout of UBAP2L confers high level resistance to etoposide. Our results provide insights to the response of cancer cells to Top2 targeting agents. Citation Format: Yilun Sun, Karin C. Nitiss, John L. Nitiss. Regulation of proteolytic repair of Top2 covalent complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4853.