Processing of TOP2-DNA covalent complexes by the ubiquitin-proteasome system

Abstract

DNA topoisomerase II (TOP2) poisons are widely used anticancer drugs which induce cytotoxic protein-DNA crosslinks termed TOP2-DNA covalent complexes. The TOP2-DNA covalent complex is a normally transient intermediate of the TOP2 reaction mechanism, whereby an intact DNA duplex is passed through an enzymemediated double strand break (DSB) in another DNA molecule. A covalent linkage between the TOP2 active site tyrosine and each DNA end conceals the break and prevents its recognition by DNA damage response proteins. However, in the presence of a TOP2 poison the TOP2-DNA complex is stabilised, leading to the accumulation of enzyme-linked DSBs which ultimately lead to cell death. Repair of the TOP2-DNA covalent complex first requires the removal of TOP2 from DNA ends, leading to the liberation of a protein-free DSB. While a number of pathways are available for the removal of the TOP2 adduct, one mechanism involves the proteasomal degradation of TOP2. The proteasomal degradation of proteins is largely regulated through the conjugation of ubiquitin to target proteins (ubiquitination). However, the requirement for ubiquitination in the processing of TOP2-DNA covalent complexes is unclear, with conflicting studies reporting both ubiquitin-dependent and -independent mechanisms. In the current study, inhibition of ubiquitination slowed the removal of TOP2 adducts from DNA and reduced the appearance of protein-free DSBs following etoposide treatment. Inhibition of the ubiquitin-dependent AAA ATPase VCP/p97 also prevented the processing of TOP2- DNA complexes to protein-free DSBs, indicating a previously unreported role for VCP/p97 in the repair of TOP2 poison-induced DNA damage. Inhibition of the ubiquitin-proteasome system increased the growth-inhibitory effects of four clinically relevant TOP2 poisons, and may be a viable strategy for the improvement of therapy with TOP2 poisons. This work confirms a ubiquitin-dependent mechanism of TOP2- DNA complex processing by the proteasome, which may be facilitated by the unfolding and extraction of TOP2-DNA complexes by VCP/p97.