A Role for VCP/p97 in the Processing of Drug-Stabilized TOP2-DNA Covalent Complexes

Abstract

DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzymeinduced double strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2.VCP/p97 is a AAA ATPase which utilises energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A-and TOP2B-DNA complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2AX, indicative of fewer DSBs . This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes, and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. Significance statementTOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers.Better understanding how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97 This article has not been copyedited and formatted. The final version may differ from this version.

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells

“…A key finding in our work is that while MRE11 overexpression can overcome proteasome inhibition, it cannot overcome inhibition of the ATPase/segregase VCP/p97. Recent work has demonstrated that VCP/p97 is important for repairing TOP2-induced damage (Swan et al, 2021), and our results agree with those findings. Interestingly, we found that inhibition of VCP/p97 by NMS873 led to lower levels of TOP2ccs than inhibition of the proteasome using the inhibitor MG132 (Supplementary Figure S3), but that inhibition of VCP/p97 was epistatic to inhibition of the proteasome by MG132.…”

“…MRE11 upregulation fails to overcome VCP/p97 inhibition for TOP2cc processing VCP/p97 is an AAA + protein that enhances proteolysis of selected substrates both from cell membranes and within the nucleus (Meyer and Weihl, 2014). Austin and colleagues recently reported that VCP/p97 inhibition hinders the repair of TOP2ccs (Swan et al, 2021). Similar to their results, we found that the VCP/p97 inhibitor NMS873 (Magnaghi et al, 2013) plus etoposide resulted in elevated levels of TOP2ccs compared to etoposide alone (Supplementary Figures 3A-D).…”

Nitrogen Mustard Induced Protein Influx in Nucleus and Metabolism Change and p97 Mediated the Repair

“…6a), which partially offset the in uence on replication pressure and DSB formation (Fig. 6b, 6C) (Swan et al, 2021). As time went on, the p97 deletion led to a large number of protein aggregation in the nucleus, and DPC could not be cleared.…”

TOP2B's contributions to transcription

“…1). In this case, the resulting TOP2-DNA covalent complexes are processed through one of a number of pathways to a protein-free state before DNA repair (59)(60)(61)(62). Classically, this occurs when cells are exposed to TOP2 poisons such as etoposide, but the enzyme can also be poisoned when it encounters endogenous DNA damage such as base excision repair (BER) intermediates (63) .…”