Effect of TDP2 on the Level of TOP2-DNA Complexes and SUMOylated TOP2-DNA Complexes.

Ka Lee,Ian Cowell,Kay Padget,Zbyslaw Sondka,Rebecca Swan,Caroline Austin

doi:10.3390/ijms19072056

Abstract

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5′-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5′-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5′ tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.

Highlights

DNA topoisomerase II (TOP2) mediates important changes in DNA topology through the induction of a double strand DNA break (DSB), followed by the passage of another intact DNA molecule through the break
After the initial proteolytic cleavage of TOP2, tyrosyl DNA phosphodiesterase 2 (TDP2) is required for the conversion of the remaining 5 -phosphotyrosine or phosphotyrosyl peptide to a 5 -phosphate, rendering the DNA end ligatable for NHEJ repair
It is generally accepted that additional factors are required for the removal of TOP2-DNA complexes by TDP2, including the proteasome and the ZATT SUMO E3 ligase

Summary

Introduction

DNA topoisomerase II (TOP2) mediates important changes in DNA topology through the induction of a double strand DNA break (DSB), followed by the passage of another intact DNA molecule through the break. To assay the activities that might remove TOP2 from TOP2-DNA covalent complexes, slides bearing “ghosts” from etoposide-treated cells were incubated with catalytically active recombinant TDP2 protein and after washing, remaining TOP2 immunofluorescence was quantified. While the recombinant TDP2 was active in the in vitro oligonucleotide assay, TDP2 did not remove TOP2A protein from genomic DNA (p = 0.1020, Figure 1D) or TOP2B (data not shown). To investigate the role of TDP2 in the proteasome-independent processing of TOP2-DNA complexes to protein-free DSBs, control or TDP2 knockdown cells were treated with 100 μM etoposide alone or in combination with 10 μM MG132 for 2 h. The level of etoposide-induced GH2AX signal was significantly reduced in the presence of MG132, reflecting the role of the proteasome in the removal of TOP2-DNA complexes and processing to DSBs (Figure 4D,E) [8,10]. Levels of remaining SUMOylated TOP2 complexes were unaffected by TDP2 knockdown, suggesting that TDP2 is not required for the proteasome-independent removal of SUMOylated TOP2-DNA complexes

Discussion

TDP2 Activity Assay

TDP2 siRNA

Findings

Western Blotting