TDP1 overexpression in human cells counteracts DNA damage mediated by topoisomerases I and II.
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.
Barthelmes
Hans U
HU
Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Medical School, Moorenstrasse 5, D-40225 Düsseldorf, Germany.
Habermeyer
Michael
M
Christensen
Morten O
MO
Mielke
Christian
C
Interthal
Heidrun
H
Pouliot
Jeffrey J
JJ
Boege
Fritz
F
Marko
Doris
D
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002166:Camptothecin / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000737:chemistry; D005047:Etoposide / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors
D001426:Bacterial Proteins; D004275:DNA, Ribosomal; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D008769:Methylnitronitrosoguanidine; D049452:Green Fluorescent Proteins; D005047:Etoposide; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C122147:tyrosyl-DNA phosphodiesterase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D002166:Camptothecin