PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

Abstract

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

Targeting DNA-Protein Crosslinks via Post-Translational Modifications

“…This finding coincided with the discovery in yeast of the first DPC protease, Wss1 (Stingele et al, 2014). (Liu et al, 2021;Krastev et al, 2022) Pli1 SUMO-targeted ubiquitylation pathway TOP1-DPC (Steinacher et al, 2013) Nse2 SUMO-targeted ubiquitylation pathway TOP1-DPC (Heideker et al, 2011) PARylation PARP1 PARP1 auto-PARylation limits PARP1 trapping, recruits PTUbLs or deubiquitylation enzymes PARP1-trapping (Prasad et al, 2014;Steffen et al, 2016;Gatti et al, 2020;Kruger et al, 2020) TOP1-DPC (Sun et al, 2021) PTM Readers/Effectors Ubiquitylation Proteasome DPC proteolysis M.HpaII-DPC (Larsen et al, 2019) DNMT1-DPC (Liu et al, 2021) TOP1-DPC (Desai et al, 1997;Mao et al, 2000b;Desai et al, 2003;Zhang et al, 2004;Lin et al, 2008;Sordet et al, 2008;Kerzendorfer et al, 2010;Heideker et al, 2011;Steinacher et al, 2013;Nie et al, 2017;Sun et al, 2020;Sun et al, 2021) TOP2-DPC (Mao et al, 2001;Wei et al, 2017;Sun et al, 2020) TOP3B-DPC (Saha et al, 2020) Polβ-DPC (Quinones et al, 2015;Quinones et al, 2020) PARP1-trapping (Prasad et al, 2019;Gatti et al, 2020) Flp-DPC (Ma et al, 2019) HMCES-DPC (Mohni et al, 2019) SPRTN a DPC proteolysis M.HpaII-DPC (Larsen et al, 2019) TOP1-DPC (Vaz et al, 2016;Maskey et al, 2017) TOP2-DPC (Lopez-Mosqueda et al, 2016;…”

“…While several studies initially reported that TOP1-DPCs are PARylated by PARP1 in vitro , the outcome of this PARylation in cells was largely unknown ( Malanga and Althaus, 2004 ; Yung et al, 2004 ; Park and Cheng, 2005 ). A recent work showed that TOP1-PARylation exhibits an adverse effect on TOP1-DPCs compared to its effect on PARP1 trapping ( Sun et al, 2021 ). By inhibiting poly (ADP-ribose) glycohydrolase (PARG), sustained PARylation on TOP1-DPCs was shown to recruit the deubiquitylating enzyme USP7 to remove ubiquitylation on TOP1-DPCs, thus preventing their proteasomal degradation ( Sun et al, 2021 ) ( Figure 1I ).…”

Human topoisomerases and their roles in genome stability and organization

“…Nucleic-acid excision pathways for TOP1 and/or TOP2 include excision by the endonucleases MRE11, CtIP and XPF–ERCC1, or excision by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 (step 4). b | TDP1 is activated by poly(ADP-ribose) polymerase 1 (PARP1) 268 , 269 , 275 , and upon cleaving DNA leaves a 3′-phosphate that is further processed by polynucleotide kinase phosphatase (not shown). TDP2 leaves a 5′-phosphate that can be directly ligated or extended by DNA polymerases (not shown).…”

“… 265 ). TDP1 activity is also controlled by its SUMOylation 266 , its phosphorylation by ATM and DNA-PK 267 , and its recruitment by poly(ADP-ribose) polymerase 1 (PARP1) 268 , 269 . ATM-deficient mice exhibit endogenous accumulation of TOP1-DPCs and neurodegeneration 250 .…”

Ribosome ADP‐ribosylation: A mechanism for maintaining protein homeostasis in cancers

“…For example, studies have shown that PARylation mediates DNA repair by reshaping chromatin into an open conformation (Poirier et al, 1982). In addition, PARylation recruits USP7, a deubiquitinating enzyme, to topoisomerase I DNA‐protein cross‐links (TOP1‐DPCs) and reverses ubiquitination of TOP1‐DPCs, indicating that PARylation involves in the repair of DPCs (Sun et al, 2021). Furthermore, poly(ADP‐ribose) polymerase 1 (PARP1) has been shown to prevent alkylation and oxidative damage of DNA in colorectal cancer, but it has also been shown to promote inflammation‐driven colorectal tumor progression (Dörsam et al, 2018).…”