Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.

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. We reveal that this enzyme is TTRAP, a member of the Mg 2+ /Mn 2+ -dependent family of phosphodiesterases, and show that cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase II-induced DNA double-strand breaks. TTRAP is the first human 5'-tyrosine phosphodiesterase to be identified and we suggest this enzyme additionally be denoted tyrosyl DNA phosphodiesterase-2 (TDP2).In an attempt to identify novel human tyrosyl DNA phosphodiesterase activities, we exploited the hypersensitivity of Saccharomyces cerevisiae tdp1Δ rad1Δ double mutant cells to camptothecin (CPT), a topoisomerase I (Top1) poison that induces single-strand breaks (SSBs) with Top1 covalently linked to the 3'-terminus 3, 10 . This strain lacks not only Tdp1 but also Rad1-Rad10 nuclease, which in yeast provides an alternative (endonucleolytic) pathway for removing Top1 from 3'-termini 11,12 . We transformed this strain with a human cDNA library 3 and screened the resulting population of transformants for cellular resistance to CPT. Of six tdp1Δ rad1Δ transformants displaying wild-type levels of CPT resistance, three harboured cDNA clones encoding TDP1 and three harboured cDNA clones encoding TTRAP (TRAF and TNF receptor-associated protein), a protein of unknown function and a putative member of the Mg 2+ /Mn 2+ -dependent phosphodiesterase super-family, with the DNA repair protein AP endonuclease-1 (APE1) being its closest relative 13,14 (Fig.1a and Supplementary Fig.1).The TDP1 and TTRAP cDNA clones recovered in the genetic screen suppressed the CPT sensitivity of tdp1Δ rad1Δ cells to a similar extent (Fig. 1b & data not shown). Whilst the pACT-TTRAP clones encoded TTRAP protein that lacked eight (pACT-TTRAP-2, pACT-TTRAP-3) or twenty-two (pACT-TTRAP-1) residues from the amino-terminus (data not shown), full-length TTRAP similarly suppressed the CPT sensitivity of tdp1Δ rad1Δ (Fig. 1c, left). In contrast, human APE1 protein failed to suppress this sensitivity, suggesting that ability to complement CPT sensitivity in tdp1Δ rad1Δ cells is not a generic feature of metaldependent phosphodiesterases (Fig. 1c, left). Conversely, whereas human APE1 suppressed the sensitivity of AP endonuclease-defective apn1Δapn2Δ tpp1Δ yeast cells to methyl methanesulphonate (MMS)-induced DNA base damage, human TTRAP did not, suggesting that the impact of TTRAP in these experiments was restricted to topoisomerase-mediated DNA damage ( Fig. 1c, right). TTRAP contains four highly conserved motifs that putatively assign this protein to the metal-dependent phosphodiesterase superfamily (see Fig.1a and Supplementary Fig.1). We thus examined whether mutation of two predicted catalytic residues (Fig.1a; E152 and D262) within two of these motifs impacted on the complementation of CPT sensitivity by TTRAP. Indeed, in contrast to wild-type TTRAP protein, ne...

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