Tyrosyl-DNA Phosphodiesterase I Processing of Topoisomerase II-DNA Covalent Complexes is Dependent on Etoposide Stabilization and Tyrosyl-DNA Phosphodiesterase I N-terminal Domain

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3'- and 5'-phosphodiester linked DNA-adducts, including Topoisomerase I- and II-DNA covalent complexes (Topo1cc and Topo2cc). Utilizing the model organism Saccharomyces cerevisiae, which only expresses Tdp1 and does not contain TDP2 orthologs or activity, we investigated the conditions under which Tdp1 processes Topo2(peptide)cc in cells and examined the role of the Tdp1 N-terminal domain in resolving Topo2(peptide)cc. We replaced the transcription activator PDR1 gene with the chimeric pdr1dbd-CYC8 transcription repressor in our strain, resulting in increased sensitivity to etoposide and doxorubicin but not to mitoxantrone. Using Tdp1 catalytic histidine mutants that induce substrate-dependent toxicity, we detected that Tdp1 selectively processes etoposide stabilized Topo2(peptide)cc. In contrast, Tdp1's processing of Topo1cc is drug independent. Moreover, we observed that the yeast SCAN1-like H432R mutant induces a more severe toxic phenotype when processing etoposide-Topo2(peptide)cc than Topo1cc with or without camptothecin. Extrapolating this observation to SCAN1 cerebellar neurons, we hypothesize that the contribution of Tdp1H493R processing of Topo2β(peptide)cc trapped by e.g., oxidative damaged nucleotides, is potentially more substantial towards cerebellar atrophy and compels further investigation. Nonetheless, the observed toxic phenotypes induced by Tdp1 mutants are recessive and suppressed by endogenous wild-type Tdp1 by lowering 5'Tdp1(mutant)cc. Moreover, Tdp1 cannot process etoposide-Topo2cc in cells without its N-terminal domain. This suggests that in cells Tdp1's N-terminal domain plays a critical role in regulating Tdp1 activity potentially by mediating recruitment to the DNA-adduct and/or facilitating accessibility to the phosphodiester linkage within a protein/peptide-DNA complex or stabilizing of the DNA strands related to damaged nucleotide-adducts.