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. 2021 Aug 18;12(1):5010.
doi: 10.1038/s41467-021-25252-9.

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

Yilun Sun  1 , Jiji Chen  2 , Shar-Yin N Huang  3 , Yijun P Su  2 , Wenjie Wang  3 , Keli Agama  3 , Sourav Saha  3 , Lisa M Jenkins  4 , John M Pascal  5 , Yves Pommier  6
Affiliations

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

Yilun Sun et al. Nat Commun. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Identification of PARG as repair factor for TOP1-DPC by single-molecule tracking.
a Top panels: filming of TOP1-HaloTag single molecules in U2OS cells transfected with TOP1-HaloTag expression plasmid. The cells were divided into seven groups: no treatment, DMSO (2h), BTZ (10 μM, 2h), PARGi (10 μM, 2h),  CPT (100 μM, 2h), CPT + BTZ and CPT + PARGi. Middle panels: plots of tracks of TOP1-HaloTag single molecules in the top-panel films. The tracks were reconstructed in two dimensions by MATLAB analysis pipeline. The neighboring tracks are of different colors to distinguish one from the others. Bottom panels: count of jumps of TOP1-HaloTag single molecules derived from the top-panel films. The X-axis of the histogram is the jump distance of TOP1 single molecules in the top-panel films and the Y-axis is the count of the jumps. The bin size is 0.1 μm. For example, the “before treatment” sample has 4120 jumps whose distances are between 0 and 0.1 μm and 86 jumps whose distances are between 1 and 1.2 μm. The sample has a total of 19120 jumps that are the sum of the count of each bin. b Quantitation of TOP1-HaloTag single molecules shown in panel (a) using ThunderSTORM, an ImageJ plug-in. Top lines are maximum values, bottom lines are minimum values and middle lines are medians. Box limits indicate the range of the central 50%. Whiskers are upper and lower quartiles. n = 10 biologically independent samples. P value was calculated by paired Student’s t-test (two-tailed distribution). **: p = 0.0017. c Time-course of TOP1-HaloTag single molecules in U2OS cells treated with DMSO, CPT (100 μM), CPT (100 μM) + PARGi (10 μM) and CPT (100 μM) + BTZ (10 μM). Images were taken at the indicated time points. The scale bar represents 10 μm. d Quantitation of TOP1-HaloTag single molecules shown in panel (c). n = 2 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value comparing TOP1 single molecules of CPT and CPT + PARGi was calculated by paired Student’s t-test (two-tailed distribution). *: p = 0.038.
Fig. 2
Fig. 2. Inhibiting PARG reveals the otherwise transient PARylation of TOP1-DPC and stabilizes TOP1-DPCs.
a His-tag pulldown assay showing reversible PARylation of transfected His-TOP1. Following transfection of 6×His-tagged TOP1 expression construct, HEK293 cells were treated with the indicated drugs for His-tag pulldown using Ni-NTA agarose under denaturing conditions. The pulldown samples and input samples were subjected to IB using α-TOP1 and α-PAR antibodies. CPT: 20 µM, 30 min; PARPi: 10 µM, 1 h pre-treatment; PARGi: 10 µM, 1 h pre-treatment. b Proximity ligation assay (PLA) showing TOP1-PAR interaction in cells treated with CPT and PARGi. Following transfection of 6×His-tagged TOP1 expression construct, U2OS cells were treated with the indicated drugs for PLA using rabbit α-His-tag antibody and mouse α-PAR antibody (10H). The scale bar represents 3 μm. c PARGi revealed CPT-induced PAR polymers on chromatin. After pre-extraction, U2OS cells treated as indicated were subjected to IF using α-PAR antibody (10H). CPT: 20 µM; PARGi: 10 µM, 1 h pre-treatment. The scale bar represents 10 μm. d Scheme of immunodetection of TOP1-DPC PARylation in vivo by modification of RADAR assay. TOP1-DPC PARylation was detected with α-PAR antibody (10H). e PARGi induced the hyper-PARylation and stabilization of TOP1-DPCs. HEK293 cells were treated with CPT (20 µM) in the absence or presence of PARGi (10 µM, 1 h pre-treatment). Cells were collected at the indicated time points for the modified RADAR for detection of TOP1-DPCs and their PARylation and ubiquitylation using α-TOP1, α-PAR, and α-Ub antibodies. 2 µg of digested DNA from each sample was subjected for slot-blotting using α-dsDNA antibody as a loading control. f ICE assay confirming that PARG deficiency blocked the removal of TOP1-DPCs. HEK293 cells transfected with control siRNA (siCtrl) or siRNA targeting PARG were treated with CPT (2 µM). Cells were collected at the indicated time points for ICE assay. 2 µg of digested DNA from each ICE assay sample was subjected for slot-blotting using α-TOP1 antibody or α-dsDNA antibody. g The modified RADAR assay showing that in vivo TOP1-DPC PARylation by PARP1 was counteracted by PARG. After transfection of the PARP1-FLAG expression plasmid, HEK293 cells were pre-treated with PARPi (10 µM, 1 h), PARGi (10 µM, 1 h), or PARPi + PARGi, followed by CPT treatment (20 µM, 1 h). The cells were then subjected to the modified RADAR assay for detection of TOP1-DPCs and their PARylation using α-TOP1 and α-PAR antibodies. h Inhibiting replication or transcription did not affect TOP1-DPC PARylation. HEK263 cells were pre-treated PARGi (10 µM, 1 h) in the absence or presence of the replication inhibitor aphidicolin (APH, 10 µM, 1 h) or the transcription inhibitor DRB (100 µM, 1 h). Cells were co-treated with 20 µM CPT for 0, 1, or 4 h, followed by detection of TOP1-DPCs and their PARylation by the modified RADAR assay using α-TOP1 and α-PAR antibodies.
Fig. 3
Fig. 3. TOP1-PARylation enhances TDP1 recruitment without enhancing TOP1cc hydrolysis.
a TOP1-TDP1 interaction was enhanced by PARGi and inhibited by PARPi. Following transfection of 6×His-tagged TOP1 expression construct, HEK293 cells were treated with the indicated drugs before His-tag pulldown using Ni-NTA agarose under native conditions. The pulldown and input samples were Western blotted with the indicated antibodies. b Proximity Ligase Assay (PLA) confirming that inhibiting PARG enhanced TOP1-TDP1 interactions. U2OS cells were treated with CPT (20 µM, 1 h), PARGi (10 µM, 2 h), or CPT + PARGi (pre-treatment with PARGi for 1 h then co-treatment with CPT and PARGi for 1 h), followed by PLA assay. The scale bar represents 3 μm. c ICE assay showing that TDP1 failed to resolve hyper-PARylated TOP1-DPCs in vivo. HEK293 cells transfected with empty vector (EV) or TDP1-FLAG overexpression plasmid were treated with CPT (2 µM) for 1 h in the absence or presence of PARGi or BTZ. Cells were collected for ICE assay. 2 µg of digested DNA from each ICE assay sample was subjected for slot-blotting using α-TOP1 antibody or α-dsDNA antibody. d Scheme for [32P]-labeled TOP1cc preparation, PARylation, and TDP1 digestion assays. e Schematic representation of the reaction products generated by TDP1 with the DNA suicidal substrate alone, unmodified TOP1ccs, and PARylated TOP1ccs. f TDP1 acted on unmodified and PARylated TOP1-DPCs at high concentrations in vitro. Representative TDP1 assay as described in panel (d) showing the 3′-nucleosidase activity of TDP1 (left set of eight samples) and TDP1’s phosphodiesterase activity toward unmodified TOP1-DPC (middle 8 samples), which is facilitated by heat denaturation of TOP1cc but does not exhibit significantly altered  activity toward PARylated-TOP1cc (right eight samples). TDP1 concentrations: 0, 1.25, 25 and 500 nM.
Fig. 4
Fig. 4. DePARylation of TOP1-DPCs is required for their proteasomal degradation.
a. Scheme for biotinylated TOP1cc preparation, PARylation, and proteasome digestion assays. b TOP1cc proteasomal digestion assay showing that unmodified TOP1ccs (without NAD+) are fully degraded by the 26S proteasome and partially degraded by the 20S proteasome after 45 min incubation whereas PARylated TOP1ccs (+ NAD+) are refractory to proteasomal degradation. c Inhibiting PARG blocked the interactions between TOP1-DPC and the proteasome subunit PSMD14. Following transfection of PSMD14-FLAG expression plasmid, HEK293 cells were treated with the indicated drugs (1 h, 10 µM PARGi pre-treatment followed by 30 min co-treatment with 20 µM CPT) and subjected to IP using an α-FLAG antibody. The immunoprecipitates and input samples were Western blotted with the indicated antibodies. d Inhibiting PARG blocked the interactions between TOP1-DPC and the proteasome subunit PSMB5. Following transfection of PSMB5-FLAG expression plasmid, HEK293 cells were treated with the indicated drugs (1 h, 10 µM PARGi pre-treatment followed by 30 min co-treatment with 20 µM CPT) and subjected to IP using an α-FLAG antibody. The immunoprecipitates and input samples were Western blotted with the indicated antibodies. e. Model for 26 S proteasome-dependent degradation of TOP1-DPCs. f Inhibiting PARG blocked the liberation of TOP1-induced DNA breaks. Upper panels: representative images of alkaline comet assay in HEK293 cells treated with DMSO, CPT (10 µM, 2 h), PARGi (10 µM, 3 h), and CPT + PARGi (pre-treatment with PARGi for 1 h then co-treatment with CPT and PARGi for 2 h). Cells were subjected to alkaline comet assay for detection of DNA breaks. Lower panel: quantitation of tail moments using OpenComet. n = 50 biologically independent cells. P value was calculated by paired Student’s t-test (two-tailed distribution). The scale bar represents 100 μm. g Inhibiting PARG attenuated TOP1-DPC-induced DNA damage response (DDR). HEK293 cells were treated with CPT in absence of the presence of PARGi (pre-treatment for 1 h). Cells were collected at the indicated time points and subjected to non-denaturing lysis and benzonase treatment, followed by Western blotting with the indicated antibodies. h Inhibiting PARG reduced CPT-induced γH2AX foci. Upper panels: representative images of IF of γH2AX and IdU foci by iSIM. U2OS cells were synchronized in the S phase by double thymidine block, followed by IdU incorporation and PARGi treatment for 1 h. Cells were then treated with CPT (1 µM) and collected at indicated time points for IF using anti-γH2AX and anti-BrdU antibodies. Lower panels: quantitation of γH2AX foci (left) and quantitation of IdU foci (right). n = 10 biologically independent samples. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). ***: p = 0.000015. NS not significant. The scale bar represents 10 μm.
Fig. 5
Fig. 5. Inhibiting PARG triggers TOP1-DPC deubiquitylation by USP7.
a His-tag pulldown-HPLC-MS/MS showing that His-TOP1 interacted USP7 under unperturbed condition. After transfection of 6×His-tagged TOP1 expression plasmid or empty vector control (pTrex), HCT116 cells were subjected to His-tag pulldown using Ni-NTA agarose, followed by HPLC-MS. b In vitro assay showing that USP7 reversed TOP1 ubiquitylation. Recombinant TOP1 was subjected to ubiquitylation with ubiquitin, Ube1 (E1), Ubc5Hα (E2), and RNF4 (E3) for 30 min, followed by termination with EDTA and incubation with increasing concentrations of recombinant USP7 for another 30 min. Samples were Western blotted with α-Ub antibody. c His-tag pulldown assay showing that PARGi enhanced TOP1-USP7 interaction. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. His-tag pulldown was performed with Ni-NTA agarose under native conditions. Western blotting was performed with the indicated antibodies. d PLA assay showing TOP1-USP7 interaction in PARGi-treated cells. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. PLA assays were performed using rabbit α-His-tag antibody and mouse α-FLAG tag antibody. The scale bar represents 10 μm. e Inhibiting USP7 restored TOP1-DPC ubiquitylation in the presence but not in the absence of PARGi. Upper panel: HEK293 cells were treated as indicated: CPT (20 µM, 1 h), CPT + FLAG-USP7 transfection, CPT + USP7i (10 µM, 1 h pre-treatment), CPT + PARGi (10 µM, 1 h pre-treatment), CPT + PARGi + FLAG-USP7 transfection, CPT + PARGi + USP7i. Following treatments, cells were subjected to the modified RADAR assay for detection of TOP1-DPCs and their ubiquitylation using α-TOP1 and α-Ub antibodies. Lower panel: densitometric quantitation of ubiquitylated TOP1-DPC signals generated from triplicate experiments including representative blots shown in (c) using ImageJ. n = 3 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). *: p = . NS not significant. f Inhibiting USP7 did not impact the induction of γH2AX upon exposure to CPT. U2OS cells were synchronized in the S phase by double thymidine and treated with CPT (1 µM) in the absence of presence of USP7i (10 µM, 1 h pre-treatment) and collected for IF by iSIM using an anti-γH2AX antibody. The scale bar represents 10 μm.
Fig. 6
Fig. 6. Model for the role of PARG in the repair of TOP1-DPCs.
a PARP1 and TOP1 form cellular protein complexes (present study and refs. ,). b TOP1-DPC trapped by CPT is rapidly modified with PAR by PARP1 and with ubiquitin (by RNF4 and potentially other E3 ligases, not shown). The PARylation recruits TDP1, PARG, and USP7 to the TOP1-DPC. c TOP1-DPC PARylation is readily and rapidly reversed by PARG, enabling the 26S proteasome to target the ubiquitylated TOP1-DPC for degradation. d TDP1 hydrolyzes the TOP1 peptide to expose the DNA ends for repair. e In the presence of PARGi, TOP1-DPC dePARylation is blocked and the persistent PAR polymers on TOP1-DPC obstruct the proteasome hence stabilize TOP1-DPC. f The stabilization of TOP1-DPC triggers USP7 to deubiquitylate the DPC to recycle the ubiquitin molecules.
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