Tyrosyl-DNA phosphodiesterase 2 (TDP2)

The process of removing torsional stress from DNA by topoisomerase II (TOP2) generates transient double-strand breaks (DSBs), 'cleavage complexes' (TOP2cc), within which the topoisomerase is covalently linked to the 5′-termini via 5′ phosphotyrosyl bonds. TOP2 usually reseals the DSB at the end of each catalytic cycle but under some conditions the cleavage complex becomes abortive, generating a potentially pathogenic DSB that requires repair. This can happen for diverse reasons including the presence of ribonucleotides incorporated into DNA, the stabilisation of cleavage complexes by anticancer drugs (e.g. etoposide, doxorubicin and mitoxantrone) and even dietary flavonoids may also stabilise Top2cc.

The enzyme 5′-tyrosyl DNA phosphodiesterase 2 (TDP2) can initiate the repair of TOP2cc by hydrolytic removal of trapped topoisomerase peptides from the 5′-termini of DSBs through hydrolysis of a 5'-phosphodiester bond, giving rise to DNA with free 5' phosphate.termini that can then be repaired.

The importance of TDP2 is illustrated by its suggested activity in resistance to chemotherapeutic topoisomerase agents such as etoposide. Genetic deletion of TDP2 results in specific and high sensitivity to the anticancer agent etoposide in avian DT40 cells, identifying TDP2 as a critical component of the cellular defence against Top2-induced DNA damage (1). It has been found overexpressed in lung cancer development (2) and, in addition, it has been shown that TDP2 protects mouse cells from cytotoxic and clastogenic effects of TOP2 poisons, further emphasising that the development of small molecule inhibitors for TDP2 may provide a way of sensitising types of tumours to chemotherapy agents (3).

These assay kits are used to follow the activity of the enzyme TDP2. The assay follows the phosphotyrosyl hydrolysing activity of human TDP2 using a fluorescently-tagged phosphotyrosine oligo (5’-(6-FAM NHS)(5’-pTyr)GATCT(3’-BHQ-1)-3’ as the substrate. The black hole quencher BHQ quenches the fluorescence of the FAM. Cleavage of the substrate by the TDP2 leads to an increase in fluorescence.

1.         Zeng Z, Cortés-Ledesma F, El Khamisy SF, Caldecott KW. TDP2/TTRAP is the major 5′-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage. Journal of Biological Chemistry. 2011 Jan 7;286(1):403-9.

2.         Li C, Fan S, Owonikoko TK, Khuri FR, Sun SY, Li R. Oncogenic role of EAPII in lung cancer development and its activation of the MAPK–ERK pathway. Oncogene. 2011 Sep;30(35):3802-12.

3.         Gómez-Herreros F, Romero-Granados R, Zeng Z, Alvarez-Quilon A, Quintero C, Ju L, Umans L, Vermeire L, Huylebroeck D, Caldecott KW, Cortés-Ledesma F. TDP2–dependent non-homologous end-joining protects against topoisomerase II–induced DNA breaks and genome instability in cells and in vivo. PLoS genetics. 2013 Mar 7;9(3):e1003226.