title: Tyrosyl-DNA-phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes in human cells.
abstract: Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3' DNA complexes, 3' phosphoglycolates, alkylation damage-induced DNA breaks, and 3' deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells.
authors.affiliation: Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Borda
authors.affiliation: Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
authors.forename: Micaela
authors.initials: M
authors.lastname: Palmitelli
authors.affiliation: Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Verón
authors.affiliation: Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
authors.forename: Marcela
authors.initials: M
authors.lastname: González-Cid
authors.affiliation: Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina. Electronic address: mnebel@hematologia.anm.edu.ar.
authors.forename: Marcelo
authors.initials: M
authors.lastname: de Campos Nebel
mesh_terms: D000951:Antigens, Neoplasm / Q000633:toxicity*; D002867:Chromones; D003114:Colony-Forming Units Assay; D004249:DNA Damage / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics* / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000633:toxicity*; D004268:DNA-Binding Proteins / Q000633:toxicity*; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005840:Gentian Violet; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015162:Micronucleus Tests; D009025:Morpholines; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C479235:2-(morpholin-4-yl)benzo(h)chromen-4-one; D000951:Antigens, Neoplasm; D002867:Chromones; D017931:DNA Primers; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009025:Morpholines; D000075223:Poly-ADP-Ribose Binding Proteins; D005047:Etoposide; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human; D005840:Gentian Violet
keywords: DNA double strand break repair; Genome instability; Topoisomerase II-mediated DNA damage; Tyrosyl-DNA-phosphodiesterase I