Abstract 3500: Nucleolytic processing of Topoisomerase 2 covalent complexes

Abstract

Abstract The generation of elevated levels of enzyme: DNA covalent complexes is the key event in cell killing by many drugs targeting DNA topoisomerases. These agents, termed topoisomerase poisons generate protein linked DNA strand that block transcription and replication, leading to cell death. A critical step in the repair of topoisomerase mediated DNA damage is the removal of protein that is covalently attached to DNA. Several specialized repair enzymes, including Tdp1 (tyrosyl DNA phosphodiesterase I) and TTRAP (TRAF and TNF receptor-associated protein) can hydrolyze phosphotyrosyl: DNA linkages. We previously reported that yeast Tdp1 could hydrolyze 5′ as well as 3′ phosphotyrosyl linkages. Human Tdp1 can also hydrolyze 5′ phosphotyrosyl linkages, although the efficiency of the reaction with the human enzyme is much less than that seen with the yeast enzyme. Interestingly, the human enzyme processes adducts bearing a seven amino acid peptide linked to an oligonucleotide with greater efficiency than a 5′ biotin linked oligonucleotide, suggesting that the nature of the adduct at DNA ends influences Tdp1 reaction kinetics. We also used substrates derived from Top2 trapped covalent complexes to assess the ability of other DNA repair enzymes to remove peptides covalently bound to DNA. We found that the heterodimeric nuclease Slx1/Slx4 is able to remove Top2 peptides that are covalently bound to DNA. This result is consistent with our genetic data from yeast that a mutation in the subunit that includes the nuclease (Slx1) is hypersensitive to Top2 poisons, but not sensitive to other DNA damaging agents. Our results indicate that there are multiple pathways for repairing Top2 covalent complexes and suggest that the Slx1/Slx4 dependent pathway may be particularly relevant to repairing topoisomerase mediated damage at replication forks. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3500.