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

Jeffrey J Pouliot,Fritz Boege,Doris Marko,Hans U Barthelmes,Morten O Christensen,Michael Habermeyer,Heidrun Interthal,Christian Mielke

doi:10.1074/jbc.m405042200

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 DNA.topoisomerase 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.

Highlights

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3؅-phosphate of DNA breaks
It should be noted that a population of transgenic cell clones with much higher expression levels of GFP-TDP1 could be selected by a combination of puromycin and camptothecin (Fig. 1A, bottom), suggesting that TDP1 overexpression counteracts topoisomerase I (topo I)-mediated cell killing, which supports recent data obtained with yeast TDP1 [23]
Our report provides for the first time information about the localization and mobility of human TDP1 in living human cells

Summary

Introduction

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3؅-phosphate of DNA breaks. 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 mammalian DNA repair, we have here overexpressed GFP chimera of human TDP1 or its inactive mutant TDP1H263A [14] in human cells and studied the effects on the outcome of various types of DNA damage

Methods

Results

Conclusion