A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

Abstract

HIGHLIGHTS• Abortive topoisomerase I (TOP1) and II (TOP2) cleavage complexes resulting in DNAprotein crosslinks (TOP-DPCs) are rapidly and sequentially modified by SUMO-2/3, SUMO-1 and ubiquitin before their proteasomal degradation. • PIAS4 SUMOylates TOP-DPCs via its DNA-binding SAP domain independently of DNA transactions and DNA damage responses. • RNF4 ubiquitylates SUMOylated TOP-DPCs and drives their proteasomal degradation.• TOP-DPC processing by the SUMO-Ub pathways is conserved in yeast and human cells. SUMMARYTopoisomerase cleavage complexes (TOPccs) can be stalled physiologically and by the anticancer drugs camptothecins (TOP1 inhibitors) and etoposide (TOP2 inhibitor), yielding irreversible TOP DNA-protein crosslinks (TOP-DPCs). Here we elucidate how TOP-DPCs are degraded via the SUMO-ubiquitin (Ub) pathway. We show that in human cells, TOP-DPCs are promptly and sequentially conjugated by SUMO-2/3, SUMO-1 and Ub. SUMOylation is catalyzed by the SUMO ligase PIAS4, which forms a complex with both TOP1 and TOP2a and b. RNF4 acts as the SUMO-targeted ubiquitin ligase (STUbL) for both TOP1-and TOP2-DPCs in a SUMOdependent but replication/transcription-independent manner. This SUMO-Ub pathway is conserved in yeast with Siz1 the ortholog of PIAS4 and Slx5-Slx8 the ortholog of RNF4. Our study reveals a conserved SUMO-dependent ubiquitylation pathway for proteasomal degradation of both TOP1-and TOP2-DPCs and potentially for other DPCs. phosphodiesterases (TDPs) that hydrolyze the covalent tyrosine-DNA bond, with TDP1 excising TOP1-DPCs (Pouliot et al., 1999) and TDP2 excising TOP2-DPCs in higher eukaryotes (Cortes Ledesma et al., 2009). In additions to TDPs, endonucleases such as Mre11 and XPF have been reported to play a key role in removing both TOP-DPCs presumably by incising the DNA adjacent to the TOP-DPC (Aparicio et al., 2016;Pommier et al., 2016;Sasanuma et al., 2018). Proteolytic degradation is required for TDP activities and earlier work showed that the ubiquitin (Ub)proteasome pathway (UPP) plays an important role in proteolyzing TOP-DPCs (Desai et al., 1997;Mao et al., 2001). Yet, the detailed molecular mechanisms by which the UPP targets TOP-DPCs are still poorly understood. In addition, small ubiquitin-like modifiers (SUMO) 1 and 2/3 have been implicated in TOP-DPC repair (Agostinho et al., 2008;Kanagasabai et al., 2009;Mao et al., 2000a;Mao et al., 2000b), but the functions of these modifications still remain elusive.SUMOylation has been linked to ubiquitylation, as polymeric SUMO chains can serve as mark for recognition by SUMO-targeted ubiquitin ligases (STUbLs) that attach Ub moieties on poly-SUMOylated substrates to promote their proteasomal degradation (Poulsen et al., 2013;Tatham et al., 2008;van Hagen et al., 2010). SUMO-Ub pathways orchestrate the proteolytic turnover of DNA damage response (DDR) and repair enzymes to facilitate DNA repair in mammalian cells (Jackson and Durocher, 2013;Ulrich, 2012). The Siz/PIAS RING family SUMO ligases PIAS1 and PIAS4 mediate SUMOylation as an early re...

SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

“…Considering recent reports showing that Topo-1 is modified by both ubiquitin and SUMO-1 after CPT treatment (Sun et al, 2019), our results suggest that SPRTN cleaves SUMOylated DPCs that also contain ubiquitin moieties.…”

“…It seems plausible that both SUMO and ubiquitin co-exist on the same substrate. In fact, our in vitro cleavage experiments on Topo-1 indicate that SPRTN efficiently cleaves Topo-1 only when isolated from CPT-treated cells, a condition that induces co-modification by SUMO and ubiquitin (Sun et al, 2019). Such crosstalk between ubiquitin and SUMO could signal SPRTN ubiquitin-dependent proteolysis of SUMOylated DPCs, thus preventing unscheduled cleavage of proteins modified by only one of the two PTMs.…”

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways

“…STUbLs are described to facilitate Top1cc processing in budding, fission yeast, and mammalian cells (Nie et al, 2017;Sharma et al, 2017;Sun et al, 2019). The Slx5-Slx8 STUbL promotes repair in a pathway parallel to Tdp1 ( Figure S1F), as also observed in fission yeast (Nie et al, 2017).…”

Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

“…To determine whether the TOP3Bccs are ubiquitylated, we performed Detection of Ubiquitylated and SUMOylated TOP-DPCs [DUST assay (Sun et al, 2019)]. In this assay RADAR samples are digested with micrococcal nuclease (MNase) to release TOP3B from TOP3Bccs and ubiquitylated TOP3B are detected by SDS-PAGE and immunoblotting with anti-Ub antibody ( Figure 4I).…”

“…For the DUST assay (Sun et al, 2019), 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 CaCl2, followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Bccs and ubiquitylated TOP3Bccs.…”