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

Abstract

During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5′ phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2β covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.

RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons

“…As mentioned above, we speculate that RNF4/proteasome pathway may compete with ZATT/ZNF451-TDP2 and RAD54L2 pathways for SUMOylated TOP2ccs substrate. Following the proteolytic degradation of TOP2ccs, small peptide residues are left, which must be processed by TDP2 or other repair enzymes such as MRN/CtIP ( 19 ). TDP2-dependent hydrolysis results in a protein/peptide-free DSB that can be ligated by NHEJ without further nucleolytic processing, providing a direct mechanism for DSB repair.…”

“…TDP2dependent hydrolysis results in a protein/peptide-free DSB that can be ligated by NHEJ without further nucleolytic processing, providing a direct mechanism for DSB repair. Nucleases like MRN/CtIP can cleave DNA strands in the vicinity of either an intact trapped TOP2cc or proteolytic degraded TOP2cc (14)(15)(16)(17)(18)(19), which generates DSB that can be further repaired by HR or NHEJ, which is potentially mutagenic. As previously reported (7), RNF4 and proteasome processing of TOP2ccs would lead to irreversible DPC formation, activate DNA damage responsive pathway, and result in cytotoxic and genome instability due to error-prone repair of these TOP2ccs intermediates.…”

“…In this case, ZATT/ ZNF451 promotes both the accessibility and activity of TDP2 toward the TOP2-DNA covalent bonds (13). On the other hand, nucleolytic enzymes involved in DSB repair, like MRE11-RAD50-NBS1 (MRN) and C-terminal binding protein 1 (CtBP1) interacting protein (CtIP), can also cleave DNA strands in the vicinity of a trapped TOP2cc, releasing the entire TOP2 DNA-protein complex (14)(15)(16)(17)(18)(19). Once trapped TOP2 proteins are removed, protein-free DNA breaks can then be recognized and repaired by major DSB repair pathways such as nonhomologous end joining (NHEJ) or homologous recombination (HR) (20).…”

The Multiple Faces of the MRN Complex: Roles in Medulloblastoma and Beyond

“…Importantly, topoisomerase activity generates DNA breaks as normal intermediates during chromosome metabolism. MRN complex prevents topoisomerase-mediated DSBs by removing TOP1 and TOP2 trapped on DNA [34][35][36][37].…”

Editorial: The repair of DNA–protein crosslinks

“…Our work in this Research Topic shows that the MRN complex can remove TOP2-DPCs by using its endonuclease activity independently of the proteasomal degradation of the DPCs. Surprisingly, the removal of TOP2-DPCs by the MRN complex was found to require the unfolding activity of VCP/p97, suggesting that unfolding of TOP2-DPCs allows the loading of the MRN repair machinery on the DNA fragment adjacent to the DPCs and hence the incision of the DNA (Sun et al, 2022).…”