Fig 1: PARP10 recruitment to ssDNA gaps and the subsequent TLS-mediated gap filling requires both the PCNA interaction ability and the catalytic activity.a. Schematic representation of the domain organization of PARP10, indicating the catalytic site mutant (G888W) and the PCNA interaction-deficient (ΔPIP) mutant used. b. Western blots showing the exogenous re-expression of wildtype, G888W catalytic site mutant, and ΔPIP PARP10 variants in PARP10-knockout HeLa cells. c, d SIRF experiments showing that PARP10 G888W catalytic site mutant and ΔPIP mutant show reduced binding to ssDNA gaps induced by treatment with 150 µM cisplatin (c) or 0.4 mM HU (d) compared to wildtype control. At least 68 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. e, f BrdU alkaline comet assays showing that re-expression of wildtype PARP10 suppresses the accumulation of replication-associated ssDNA gaps upon treatment with 150 µM cisplatin (e) or 0.4 mM HU (f) in HeLa PARP10-knockout cells, but re-expression of the G888W catalytic site mutant or of the ΔPIP mutant does not. At least 49 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. g, h S1 nuclease DNA fiber combing assays showing that re-expression of wildtype PARP10 suppresses the accumulation of nascent strand ssDNA gaps upon treatment with 150 µM cisplatin 0.4 mM HU in HeLa PARP10-knockout cells, but re-expression of the G888W catalytic site mutant or of the ΔPIP mutant does not. Quantifications (g) and representative micrographs, with scale bars representing 10 µm (h) are shown The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 70 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. i SIRF experiments showing that re-expression of wildtype PARP10 promotes the binding of RAD18 to ssDNA gaps induced by treatment with 0.4 mM HU in HeLa PARP10-knockout cells, but re-expression of the G888W catalytic site mutant or of the ΔPIP mutant does not. At least 72 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. j Co-immunoprecipitation experiment in HeLa cells showing the interaction of PARP10 variants with RAD18. k PLA assay showing the co-localization between RAD18 and PARP10 variants upon treatment with 0.4 mM HU for 3hrs in HeLa cells. The catalytic site mutant (G888W) shows similar RAD18 co-localization as the wildtype form, while the PCNA interaction-deficient (ΔPIP) mutant shows reduced RAD18 co-localization. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). l SIRF experiments showing that re-expression of wildtype PARP10 promotes the binding of REV1 to ssDNA gaps induced by treatment with 0.4 mM HU in HeLa PARP10-knockout cells, but re-expression of the G888W catalytic site mutant or of the ΔPIP mutant does not. At least 72 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Source data are provided as a Source Data file.
Fig 2: Schematic representation of the proposed models.PRIMPOL-derived ssDNA gaps can be filled through BRCA-mediated homology-dependent repair, or through PCNA ubiquitination-mediated TLS. Our work shows that PARP10 promotes the recruitment of RAD18, the main ubiquitin ligase for PCNA. Upon PCNA ubiquitination, TLS polymerases such as REV1 are recruited for gap filling. In cells with concomitant inactivation of BRCA and PARP10, ssDNA gaps are expanded by the MRE11 exonuclease, resulting in cytotoxicity. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
Fig 3: PARP10 binds to nascent strand gaps and promotes the recruitment of RAD18 to these structures.a–c SIRF experiments showing that treatment with 150 µM cisplatin (a) or 0.4 mM HU (b, c) induces binding of PARP10 to nascent DNA in HeLa cells. Quantifications (a, b) and representative micrographs, with scale bars representing 10 µm (c) are shown. At least 76 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. d Co-immunoprecipitation experiment in HeLa cells showing that PARP10 co-precipitates with RAD18. Cells were treated with 0.4 mM HU, 150 µM cisplatin, or left untreated as indicated. e–g PLA assays showing that RAD18 and PARP10 co-localize upon treatment with 0.4 mM HU in HeLa cells. Knockdown of PARP10 or RAD18 is used as control to confirm the specificity of the PLA signals observed. Quantifications (e, f) and representative micrographs, with scale bars representing 10 µm (g) are shown. At least 45 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). h–j SIRF experiments showing that PARP10 depletion reduces the binding of RAD18 to nascent DNA upon treatment with 150 µM cisplatin (h) or 0.4 mM HU (i, j) in HeLa cells. Quantifications (h, i) and representative micrographs, with scale bars representing 10 µm (j) are shown. At least 70 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Source data are provided as a Source Data file.
Fig 4: Mono-ADP-ribosylation of RAD18 by PARP10.a In vitro ADP-ribosylation enzymatic assays showing that PARP10 can MARylate RAD18. Recombinant proteins were incubated with biotin-NAD+ and substrate MARylation is detected using streptavidin-HRP blots. RAD18 and Flag (PARP10) blots are presented to show loading controls. b–d PLA assay showing that loss of PARP10 reduces the co-localization between RAD18 and mono-ADP-ribose (MAR) upon treatment with 0.4 mM HU for 3hrs in HeLa cells. Single antibody (MAR only) control is shown to confirm the specificity of the PLA signals observed. Quantifications (b, c) and representative micrographs, with scale bars representing 10 µm (d) are shown. At least 49 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). e PLA assay showing the impact of PARP10 mutations on the co-localization between RAD18 and mono-ADP-ribose. Expression of wildtype PARP10, but not of the G888W catalytic site mutant or the PIP-box mutant restored the MAR-RAD18 PLA signal in PARP10KO cells. At least 60 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Source data are provided as a Source Data file.
Fig 5: Loss of PARP10 suppresses PCNA ubiquitination and REV1 recruitment to nascent strand gaps.a–c SIRF experiments showing that PARP10 depletion, deletion or inhibition reduces the levels of ubiquitinated PCNA at nascent DNA gaps induced by treatment with 0.4 mM in HeLa cells. Quantifications (a, b) and representative micrographs, with scale bars representing 10 µm (c) are shown. At least 53 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming knockdowns of RAD18 and USP1, which are used as controls, are shown in Supplementary Fig. 1c, d. d–f SIRF experiments showing that PARP10 depletion reduces the binding of REV1 to nascent DNA upon treatment with 150 µM cisplatin (d) or 0.4 mM HU (e, f) in HeLa cells. Quantifications (d, e) and representative micrographs, with scale bars representing 10 µm (f) are shown. At least 63 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. g, h. BrdU alkaline comet assays showing that PARP10 depletion causes accumulation of replication-associated ssDNA gaps upon treatment with 150 µM cisplatin (g) or 0.4 mM HU (h) in wildtype, but not in RAD18-knockout HeLa cells. At least 73 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. Western blots confirming RAD18 knockout are shown in Supplementary Fig. 1e. Source data are provided as a Source Data file.
Supplier Page from Abcam for Recombinant Human RAD18 protein (GST tag N-Terminus)