Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase 1 DNA-protein crosslinks and 3'-blocking lesions in the absence of tyrosyl-DNA phosphodiesterase 1 (TDP1).

Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1-DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3'-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide-DNA complex, respectively. Although TDP1-deficient (TDP1-/-) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3' -blocking lesions. These experiments showed that TDP1-/-TDP2-/- cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1-/- or TDP2-/- cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3'-blocking lesions.

Repair of trapped topoisomerase II covalent cleavage complexes: Novel proteasome-independent mechanisms.

Topoisomerase II (TOP2) resolves topologically entwined duplex DNA. It generates a transient DNA double-strand break intermediate, known as TOP2 cleavage complex (TOP2cc) that contains a covalent link between TOP2 and the 5'-terminus of the incised DNA duplex. Etoposide, a frontline anticancer drug, freezes the intermediate and forms irreversible TOP2ccs. Tyrosyl-DNA phosphodiesterase 2 (TDP2) is thought to repair irreversible TOP2ccs by hydrolyzing the phosphodiester bond between TOP2 and DNA after the proteasomal degradation of trapped TOP2ccs. However, the functional cooperation between TOP2 and proteasome in the repair of trapped TOP2ccs in vivo remains unknown. In this study, we analyze the repair of etoposide-induced TOP2ccs in wild-type and TDP2-deficient (TDP2-/-) TK6 cells in the absence and presence of MG132, a potent proteasome inhibitor. The results suggested that TOP2ccs were repaired by proteasome-dependent and proteasome-independent pathways. Both proteasome-dependent and proteasome-independent pathways were further subdivided into TDP2-dependent and TDP2-independent pathways, indicating that four pathways operate in the repair of TOP2ccs. In cell survival assays, MG132 increased the etoposide sensitivity of TDP2-/- cells, supporting the TDP2-independent and proteasome-dependent pathway among these multiple repair pathways. We also demonstrated that TDP2 released TOP2 from DNA that contained etoposide-induced TOP2cc without proteolytic degradation in vitro. Taken together, the present findings uncover novel proteasome-independent mechanisms for the repair of TOP2ccs.