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A human 5′-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage

Abstract

Topoisomerases regulate DNA topology and are fundamental to many aspects of chromosome metabolism1,2. Their activity involves the transient cleavage of DNA, which, if it occurs near sites of endogenous DNA damage or in the presence of topoisomerase poisons, can result in abortive topoisomerase-induced DNA strand breaks3,4,5. These breaks feature covalent linkage of the enzyme to the DNA termini by a 3′- or 5′-phosphotyrosyl bond and are implicated in hereditary human disease6,7,8, chromosomal instability and cancer4,9, and underlie the clinical efficacy of an important class of anti-tumour poisons3,9,10. The importance of liberating DNA termini from trapped topoisomerase is illustrated by the progressive neurodegenerative disease observed in individuals containing a mutation in tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that cleaves 3′-phosphotyrosyl bonds6,7,8. However, a complementary human enzyme that cleaves 5′-phosphotyrosyl bonds has not been reported, despite the effect of DNA double-strand breaks containing such termini on chromosome instability and cancer6,7,8. Here we identify such an enzyme in human cells and show that this activity efficiently restores 5′-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. This enzyme, TTRAP, is a member of the Mg2+/Mn2+-dependent family of phosphodiesterases. Cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase-II-induced DNA double-strand breaks. TTRAP is, to our knowledge, the first human 5′-tyrosyl DNA phosphodiesterase to be identified, and we suggest that this enzyme is denoted tyrosyl DNA phosphodiesterase-2 (TDP2).

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Figure 1: Human TTRAP suppresses the sensitivity of yeast to Top1-induced DNA damage.
Figure 2: TTRAP possesses 3′- and 5′-tyrosyl DNA phosphodiesterase (TDP) activity.
Figure 3: Reduced 5′-TDP activity in TTRAP-deficient human cell extracts.
Figure 4: TTRAP affects etoposide sensitivity and DNA damage accumulation.

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Acknowledgements

This work was funded by the MRC (G0600776), the BBSRC (BB/C516595/1), and CR-UK (C6563/A10192). S.F.E.K. and F.C.L. were also funded by Fellowships from the Wellcome Trust (S.F.E.K.; 085284), Marie Curie (FCL; 2007-2-1-IEF-221222) and EMBO (FCL; ALTF 956-2006). We thank T. Wilson, H. Nash, M. Neale, J. Nitiss and E. Hoffmann for materials.

Author Contributions F.C.L. developed the genetic screen and conducted the mammalian cell experiments. M.C.Z. and F.C.L. conducted the yeast experiments. K.O. and S.F.E.K. prepared the recombinant proteins, and S.F.E.K. conducted the biochemical experiments. K.W.C., F.C.L. and S.F.E.K. designed and interpreted the experiments. K.W.C. coordinated the project and wrote the manuscript.

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Correspondence to Felipe Cortes Ledesma or Keith W. Caldecott.

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Ledesma, F., El Khamisy, S., Zuma, M. et al. A human 5′-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature 461, 674–678 (2009). https://doi.org/10.1038/nature08444

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