TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II

Abstract

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA⅐topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1 H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.Tyrosyl DNA phosphodiesterase 1 (TDP1) 1 is an enzyme capable of hydrolyzing phosphodiester bonds between tyrosine and the 3Ј-phosphate of DNA (1, 2), which are typically generated in a transient manner by DNA topoisomerase I (topo I) (3). In keeping with this, yeast deletion mutations of TDP1 are deficient in the repair of DNA damage induced by camptothecin, a drug that stabilizes the transient topo I⅐DNA intermediate (2, 4 -6). More precisely, TDP1 has been characterized in these studies as a non-exclusive effector upstream of Rad52 that removes structurally modified topo I adducts (7, 8) as well as oxidative adducts (9) from the 3Ј-phosphate of a DNA break prior to homologous recombination repair. In mammals, TDP1 is (in addition or instead?) involved in an XRCC-dependent single-stranded DNA repair pathway also directed at topo I⅐DNA adducts (10 -12). Despite all the evidence of yeast deletion studies implying TDP1 in DNA repair, a familial disease caused by a mutation in the active site of the human ortholog of the enzyme exhibits a phenotype not at all typical for inadequate DNA repair, namely a slow onset of neuronal degeneration (13). This unexpected finding has prompted speculations that at least in mammals TDP1 could serve a much broader scope of functions, some of which may not even depend on catalytic activity. To further clarify the importance of TDP1 for...

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…2A). This is in agreement with studies using fluorescence microscopy demonstrating localization of Tdp1 to the nucleus in human cells [26].…”

“…Yet, the effects of Top2 inhibitors on TDP1-deficient yeast cells have been contradictory [53,54]. Moreover, a single study in mammalian cells has demonstrated that overexpression of an active Tdp1 significantly reduces the DNA damage caused by Top2 inhibitors [26].…”

“…SCAN1 cells have also demonstrated enhanced sensitivity to the killing effects of CPT [28,72]. Furthermore, complementary studies have shown that overexpression of wild-type Tdp1 protects cells against CPT-induced cell death [26,73], whereas the inactive mutant Tdp1 H263A does not [26]. Taken together, these data implicate Tdp1 in the repair of Top1-mediated DNA damage and demonstrate that a single defect in Tdp1 activity in human cells is adequate for CPT hypersensitivity, which is in contrast to the conditional mutations required in TDP1-deficient yeast cells.…”

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

“…TOP1 can become covalently attached to the DNA, forming TOP1 cleavage complexes (TOP1ccs) [36], and many types of endogenous DNA modifications can cause TOP1ccs [37]. The observation that spinocerebellar ataxia with axonal neuropathy (SCAN1) results from mutations in the TDP1 gene [22], which encodes a protein involved in the repair of TOP1ccs [38,39] indicates the importance of repair of such complexes in preventing ataxia. In view of the high levels of TOP1 in Purkinje neurons, it follows that these cells would be at elevated risk for TOP1ccs, and thus the loss of ability to repair such complexes would be particularly severe in these cells.…”

The Role of Multi-Drug Resistance Associated Protein 4 and P-glycoprotein in Resistance of Neuroblastoma to Topotecan and Irinotecan

“…Increased DNA repair via tyrosyl-DNA-phosphodiesterase overexpression can also lead to camptothecin analog resistance (69). Finally, ABC transporter-mediated efflux from the cell may result in topotecan resistance.…”