Debulking of topoisomerase DNA-protein crosslinks (TOP-DPC) by the proteasome, non-proteasomal and non-proteolytic pathways

Sun, Yilun; Saha, Liton Kumar; Saha, Sourav; Jo, Ukhyun; Pommier, ﻿Yves

doi:10.1016/j.dnarep.2020.102926

Cited by 52 publications

(78 citation statements)

References 220 publications

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“…As the ubiquitin-proteasome system is a pivotal step in the degradation and repair of TOP1-DPCs 3,16,21 , we pre-treated the cells with the proteasome inhibitor bortezomib (BTZ) and found, as expected, that BTZ blocked the overall downregulation of TOP1 single molecules in response to CPT but BTZ alone did not impact either the levels or the dynamics of TOP1 single molecules (Fig. 1a, b; Supplementary Movies 4 and 7).…”

Section: Resultsmentioning

confidence: 62%

“…2e, 3rd panel from the top, compare lanes 9−14 with lanes 2−7), suggesting that PARG is required for the repair of TOP1-DPCs. Given the crucial role of the 26S proteasome system for TOP1-DPC repair 3,16 , we hypothesized that persistent PARylation likely prevents the proteasome-dependent removal of TOP1-DPCs.…”

Section: Resultsmentioning

confidence: 99%

“…TOP1-DPC are repaired by redundant pathways 3,13 regulated by post-translational modifications (PTMs); of which ubiquitylation plays a dominant role by inducing the proteasomal degradation of TOP1-DPCs 3 . TOP1-DPCs are ubiquitylated either in replication/transcription-dependent manners 14,15 or in response to SUMO modifications 16 .…”

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confidence: 99%

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

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

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

et al. 2021

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“…The antibodies that were used in this study: mouse anti-βactin (AC-15, 1:20000) and mouse anti-FLAG (F-3165, 1:7000; IF 1:300) from Sigma-Aldrich; rabbit anti-TOP1 (A302-589A,1:5000), TOP2a (A300-054A, 1:4000), TOP2b (A300-950A, 1:2000) and 53BP1(A300-272A,1:150) were from Bethyl Laboratories (Montgomery, Texas, USA); rabbit anti phosphohistone H2A.X clone 20E3 (#9718, IF:1:100) from Cell Signaling Technology (Danvers, Massachusetts); mouse monoclonal anti-ubiquitin (P4D1, sc-8017 1:1000), mouse anti-EBV-BZLF1 (sc-53904, 1:1000) and mouse anti-BRCA1 (D-9, sc-6954, IF 1:100) from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti-SUMO2/3 (8A2, ab81371, 1:2000) from Abcam (Cambridge, MA, USA); monoclonal rat anti-EBV-BPLF1 (1:1500) (van Gent et al, 2014) from the MAB core facility, Helmholtz Center, Munich, Germany; mouse anti-EBV-BMRF1(1:10000) and rat anti-EBV-BFRF3 (1:1000) from Dr. Jaap M. Middeldorp (VU University Medical Center, Amsterdam, Netherlands). Alexa Fluor anti-rabbit-488 (A31570, 1:1000) and anti-mouse-555 (A315721, 1:1000) conjugated secondary antibodies raised in donkey were from Thermo Fisher (Waltham, Massachusetts, USA).…”

Section: Antibodiesmentioning

confidence: 99%

“…While TOP2-indued DSBs are relatively frequent in genomic DNA (Morimoto et al, 2019), failure to resolve TOP2ccs, as may occur upon endogenous or chemical stress that inhibits TOP2 activity, results in the formation of stable TOP2-DNA adducts that hinder DNA replication and transcription and trigger apoptotic cell death (Kaufmann, 1998). 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).…”

Section: Introductionmentioning

confidence: 99%

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

Nagy

Liu

et al. 2021

Preprint

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Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2a and TOP2b in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc), and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2bcc, enhanced TOP2b SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.

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DNA Topoisomerase Targeting Drugs

Thomas¹,

Bates²,

Figg³

et al. 2022

Holland‐Frei Cancer Medicine

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Overview Topoisomerases are an important class of enzymes that are ubiquitously expressed across all organisms, and function to resolve deoxyribonucleic acid (DNA) supercoiling and prevent and resolve ribonucleic acid (RNA) entanglements. These biochemically distinct enzymes play a critical role in DNA replication, transcription, and cellular division. Inhibitors that target specific topoisomerases, therefore, represent a major component of the anticancer chemotherapeutic armamentarium. In this chapter, we provide an overview of topoisomerase biology and topoisomerase inhibitors. We review the common and specific mechanisms of action and the molecular pharmacology of the drugs that target the topoisomerase cleavage complexes by interfacial inhibition. Molecular pathways for cancer cell death and DNA repair associated with each specific topoisomerase inhibitor are discussed. Other mechanisms of anticancer activity of selected topoisomerase inhibitors that include DNA intercalation and/or generation of oxygen radicals destabilizing and damaging chromatin structure are described. We summarize the clinical use, toxicity profile, and the molecular basis of sensitivity and resistance to topoisomerase inhibitors. We also discuss the pharmacogenomics focusing on genetic variants in drug‐metabolizing enzymes, transporters, and other proteins associated with topoisomerase inhibitors. Evolving data demonstrate the important role of molecular profiling and analysis of genetic variants in individuals that enable personalized dosing and enhance therapeutic efficacy, minimizing toxicity. Ongoing efforts focus on optimization of treatment selection, identification of novel combination therapies, elucidation of the molecular basis of response and resistance, and development of strategies for tumor‐targeted delivery based on tumor molecular signatures.

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Debulking of topoisomerase DNA-protein crosslinks (TOP-DPC) by the proteasome, non-proteasomal and non-proteolytic pathways

Cited by 52 publications

References 220 publications

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

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

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

DNA Topoisomerase Targeting Drugs

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