A conserved SUMO pathway repairs topoisomerase DNA-protein cross-links by engaging ubiquitin-mediated proteasomal degradation

Abstract

Topoisomerases form transient covalent DNA cleavage complexes to perform their reactions. Topoisomerase I cleavage complexes (TOP1ccs) are trapped by camptothecin and TOP2ccs by etoposide. Proteolysis of the trapped topoisomerase DNA-protein cross-links (TOP-DPCs) is a key step for some pathways to repair these lesions. We describe a pathway that features a prominent role of the small ubiquitin-like modifier (SUMO) modification for both TOP1- and TOP2-DPC repair. Both undergo rapid and sequential SUMO-2/3 and SUMO-1 modifications in human cells. The SUMO ligase PIAS4 is required for these modifications. RNF4, a SUMO-targeted ubiquitin ligase (STUbL), then ubiquitylates the TOP-DPCs for their subsequent degradation by the proteasome. This pathway is conserved in yeast with Siz1 and Slx5-Slx8, the orthologs of human PIAS4 and RNF4.

DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks

“…To further establish the ubiquitination and proteasomal processing of TOP3Bccs, HCT116 cells were also transfected with R338W-TOP3B and treated with either MG132 or TAK243 before harvest. RADAR assay samples were prepared and IPed with the anti-TOP3B antibody and the levels of ubiquitinated TOP3Bccs determined as recently described for TOP1ccs and TOP2ccs ( Sun et al, 2020a ). While, as expected, MG132 increased TOP3Bcc ubiquitination, TAK243 reduced the ubiquitination of TOP3Bccs ( Figure 4G ).…”

“…It reveals the role of TRIM41 as an E3 ubiquitin ligase for TOP3Bccs. Based on the redundancy of the repair pathways for TOP1 and TOP2 cleavage complexes ( Sun et al, 2020a , 2020b , 2020c ), further studies are warranted to determine whether additional ubiquitylation, proteolytic, and endonucleolytic pathways are involved in the repair of TOP3Bccs. Finally, the self-poisoning mutant R338W-TOP3B is likely to provide a molecular tool to study and map TOP3Bccs in DNA and RNA.…”

“…For detection of ubiquitinated TOP3Bccs ( Sun et al, 2019 , 2020a ), nucleic acids and covalent protein-nucleic acid adducts were recovered from FLAG-tagged R338W-TOP3B transfected cells using the RADAR assay. 8 μg of each RADAR assay sample was digested with 250 units micrococcal nuclease (Thermo Fisher Scientific, 100 units/μl) in the presence of 5 mM CaCl 2 , followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Bccs and ubiquitinated TOP3Bccs.…”

The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication

“…SUMOylation plays multiple roles in the debulking of TOP2ccs. It may mediate TOP2 proteolysis by serving as a recognition signal for ubiquitination mediated by SUMO-targeted ubiquitin ligases such as RNF4 (Sun, Miller Jenkins, et al, 2020) or may recruit SprT-family metalloproteases, such as SPRTN (Lopez-Mosqueda et al, 2016) and ARC/GCNA (Borgermann et al, 2019), that are involved in the proteasome-independent proteolytic debulking of DNA-protein adducts. Interestingly, the activity of SPRTN is inhibited by mono-ubiquitination (Stingele et al, 2016) and yet unpublished findings suggest that failure to deubiquitinate SPRTN upon depletion of the cellular deubiquitinase USP11 leads to the accumulation of unrepaired DNA-protein adducts (Perry et al, 2020).…”

“…Thus, cellular defense mechanisms attempt to resolve the TOP2ccs via proteolytic or non-proteolytic mechanisms (Sun, Saha, et al, 2020). These may involve the displacement of TOP2 via ubiquitin (Mao et al, 2001) or SUMO and ubiquitin-dependent (Sun, Miller Jenkins, et al, 2020) proteasomal degradation, which, following the removal of residual peptide-DNA adducts by the Tyrosyl-DNA phosphodiesterase-2 (TDP2) resolving enzyme (Gao et al, 2014;Pommier et al, 2014), unmasks the DNA breaks and promotes activation of the DNA damage response (DDR) (Pommier et al, 2014). Alternatively, SUMOylation may induce conformational changes in the TOP2 dimer the expose the covalent bonds to the direct action of TDP2 (Schellenberg et al, 2017).…”