Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 1. Loss of EXO1 suppresses the accumulation of nascent strand ssDNA gaps induced by HU and cisplatin in PRIMPOL-overexpressing cells. ( A ) Western blots showing the expression of PRIMPOL in control and PRIMPOL-overexpressing HeLa and U2OS cells. ( B ) Western blots showing siRNA-mediated knockdown of EXO1 and MRE11 in HeLa-PRIMPOL OE cells. ( C–F ) BrdU alkaline comet assay showing that knockdown of EXO1 suppresses the accumulation of replication-associated ssDNA gaps induced by treatment with 150 μM cisplatin ( C, D ) or 0.4mM HU ( E, F ) in HeLa ( C, E ) and U2OS ( D, F ) PRIMPOL-o v ere xpressing cells, similar to MRE11 depletion. At least 100 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, t wo-t ailed ) . Schematic representations of the assay conditions are shown at the top. ( G, H ) S1 nuclease DNA fiber combing assa y s sho wing that knockdo wn of EXO1 suppresses the accumulation of nascent strand ssDNA gaps induced by treatment with 0.4mM HU in U2OS ( G ) and HeLa ( H ) PRIMPOL-overexpressing cells, similar to MRE11 depletion. 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 35 tracts were quantified for each sample. Asterisks indicate statistical significance ( Mann-Whitney, t wo-t ailed ) . Schematic representations of the assay conditions are shown at the top.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Western Blot, Expressing, Control, Knockdown, Alkaline Single Cell Gel Electrophoresis, MANN-WHITNEY

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 2. Loss of USP1 suppresses the accumulation of nascent strand ssDNA gaps induced by HU and cisplatin in PRIMPOL-overexpressing cells. ( A ) Western blots showing siRNA-mediated knockdown of RAD18 and USP1 in HeLa-PRIMPOL OE cells, and the impact on PCNA ubiquitination, using an antibody that specifically recognizes mono-ubiquitinated PCNA. (B–E) BrdU alkaline comet assay showing that knockdown of USP1 suppresses the accumulation of replication-associated ssDNA gaps induced by treatment with 0.4 mM HU ( B, C ) or 150 μM cisplatin ( D, E ) in HeLa ( B, D ) and U2OS ( C, E ) PRIMPOL-o v ere xpressing cells. At least 100 nuclei w ere quantified f or each condition. T he median v alues are mark ed on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitne y, tw o-tailed). Schematic representations of the assay conditions are shown at the top. (F, G) S1 nuclease DNA fiber combing assa y s sho wing that knockdo wn of USP1 suppresses the accumulation of nascent strand ssDNA gaps induced b y treatment with 0.4 mM HU in HeLa ( F ) and U2OS ( G ) PRIMPOL-o v ere xpressing cells. 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 30 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitne y, tw o-tailed). Schematic representations of the assay conditions are shown at the top.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Western Blot, Knockdown, Ubiquitin Proteomics, Alkaline Single Cell Gel Electrophoresis

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 3. USP1 depletion suppresses the binding of MRE11 and EXO1 to replication stress-induced nascent strand ssDNA gaps in PRIMPOL-o v ere xpressing cells. (A–C) SIRF experiments showing that treatment with 0.4mM HU induces binding of MRE11 to nascent DNA in HeLa ( B ) and U2OS ( C ) PRIMPOL-o v ere xpressing cells, and this binding is suppressed by USP1 depletion. The labeling scheme ( A ) is designed to capture MRE11 binding to the 3 ′ end of the gap (for simplicity, only one strand, e.g. the leading strand, is shown in the schematic representation; EdU-labeled nascent DNA is indicated in red). At least 100 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–F ) SIRF experiments showing that treatment with 0.4mM HU induces binding of EXO1 to nascent DNA in HeLa (E) and U2OS (F) PRIMPOL-o v ere xpressing cells, and this binding is suppressed by USP1 depletion. The labeling scheme (D) is designed to capture EXO1 binding to the 5 ′

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Binding Assay, Labeling, Two Tailed Test

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 4. Loss of USP1 suppresses the accumulation of nascent strand ssDNA gaps induced by HU and cisplatin in PRIMPOL-o v ere xpressing cells. ( A-D ) BrdU alkaline comet assay showing that inhibition of USP1 by treatment with ML323 as indicated, suppresses the accumulation of replication-associated ssDNA gaps induced by exposure to 150 μM cisplatin ( A, B ) or 0.4mM HU ( C, D ) in U2OS (A, C) and HeLa (B, D) PRIMPOL-o v ere xpressing cells. At least 70 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitne y, tw o-tailed). Schematic representations of the assay conditions are shown at the top. ( E ) S1 nuclease DNA fiber combing assay showing that inhibition of USP1 by treatment with ML323 as indicated suppresses the accumulation of replication-associated ssDNA gaps induced by exposure to 0.4 mM HU in HeLa PRIMPOL-o v ere xpressing cells. T he ratio of CldU to IdU tract lengths is presented, with the median v alues mark ed on the graphs and listed at the top. At least 50 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann–Whitney, t wo-t ailed). A schematic representation of the assay conditions is shown at the top. (F, G) SIRF experiments showing that USP1 inhibition by ML323 treatment as indicated suppresses the binding of MRE11 ( F ) and EXO1 ( G ) to nascent DNA in HeLa PRIMPOL-o v ere xpressing cells, similar to USP1 depletion. At least 100 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.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Alkaline Single Cell Gel Electrophoresis, Inhibition, MANN-WHITNEY, Binding Assay, Two Tailed Test

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 5. Inhibition of USP1 de-ubiquitination activity promotes TLS-mediated gap filling in PRIMPOL-o v ere xpressing cells. (A, B) BrdU alkaline comet assa y s sho wing that RAD18 depletion re v erses the suppression of ssDNA gap accumulation caused b y inhibition of USP1 in U2OS ( A ) and HeLa ( B ) PRIMPOL-o v ere xpressing cells upon treatment with cisplatin or HU as indicated. At least 100 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, t wo-t ailed). Schematic representations of the assay conditions are shown at the top. (C, D) BrdU alkaline comet assays showing that REV1 depletion reverses the suppression of ssDNA gap accumulation caused by inhibition of USP1 in HeLa PRIMPOL-overexpressing cells upon treatment with cisplatin ( C ) or HU ( D ) as indicated. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann-Whitne y, tw o-tailed). Schematic representations of the assay conditions are shown at the top. ( E ) Western blots showing siRNA-mediated knockdown of REV1 in HeLa-PRIMPOL OE cells. ( F ) S1 nuclease DNA fiber combing assay showing that depletion of REV1 partially restores HU-induced ssDNA gap accumulation in HeLa PRIMPOL-o v ere xpressing cells treated with the USP1 inhibitor ML323. 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). A schematic representation of the assay conditions is shown at the top.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Inhibition, Ubiquitin Proteomics, Activity Assay, MANN-WHITNEY, Western Blot, Knockdown, Two Tailed Test

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 6. Processing of PRIMPOL-derived ssDNA gaps by MRE11, EXO1 and USP1 leads to DSB formation. (A–D) Neutral comet assays showing that treatment with 0.4 mM HU ( A, B ) or 150 μM cisplatin ( C, D ) for 2 h causes accumulation of DSBs in U2OS (A, C) and HeLa (B, D) PRIMPOL-o v ere xpressing cells, which are suppressed by depletion of MRE11, EXO1 or USP1. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, t wo-t ailed). (E, F) Neutral comet assays showing that inhibition of MRE11 endonuclease activity using the specific inhibitor PFM01 (100 μM) suppresses DSB accumulation in PRIMPOL-o v ere xpressing HeLa cells upon treatment with 0.4mM HU ( E ) or 150 μM cisplatin ( F ) for 2 h. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, t wo-t ailed). Schematic representations of the assay conditions are shown at the top.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Derivative Assay, MANN-WHITNEY, Inhibition, Activity Assay

Journal: Nucleic acids research

Article Title: USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

doi: 10.1093/nar/gkad1237

Figure Lengend Snippet: Figure 7. Schematic representation of the proposed model. PRIMPOL-generated gaps are engaged by MRE11 and EXO1, even in BRCA-proficient cells, and expanded bidirectionally. USP1, through de-ubiquitination of PCNA, promotes gap expansion by MRE11 and EXO1, presumably by suppressing TLS-mediated gap filling. Under these conditions, upon e xtensiv e processing by MRE11 and EXO1, PRIMPOL-generated gaps can be con v erted into DSBs. Created with BioRender.com.

Article Snippet: For PRIMPOL overexpression, the pLV[Exp]-Hygro-CMV > hPRIMPOL lentiviral construct ( Cyagen ) was used.

Techniques: Generated, Ubiquitin Proteomics

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , An experimental scheme depicting how targeted CRISPR–Cas9 screens performed. Two separate screens were performed in biological triplicates utilizing pool 1 sgRNA library (1,117 genes targeted) or pool 2 sgRNA library (288 genes targeted). b , A volcano plot depicting a measure of statistical significance (−log 10 (MaGeCK score)) plotted against log 2 (fold-change) in abundance of sgRNAs targeting indicated genes in cisplatin-treated versus untreated conditions in eHAP iCas9 WT cells infected with the pool 2 sgRNA library. Labelled genes are coloured based on the DDR pathway in which they operate. c , A Venn diagram comparing genes scoring as significantly depleted in cisplatin-treated arms of the screen depicted in b performed in eHAP cells (yellow) and those in a genome-wide CRISPR–Cas9 dropout screen performed in RPE-1 p53 KO cells (blue) in ref. . d , A western blot depicting loss of PrimPol protein in an isogenic eHAP iCas9 PrimPol KO clone. e , Population doublings of eHAP iCas9 WT (grey) and PrimPol KO (red) cells plotted against time to demonstrate cell growth in the absence (squares) or presence of 450 nM (circles) or 550 nM (triangles) cisplatin. Growth curve experiments were performed in n = 2 biological replicates. Data are presented as means. f , Experimental scheme for a Cell Titer Glo viability assay to determine the cisplatin sensitivity of eHAP iCas9 WT or PrimPol KO cells complemented with WT PrimPol, PrimPol AxA (catalytic mutant Asp114Ala, Glu116Ala) or PrimPol CH (primase mutant Cys419Gly, His426Tyr). g , A western blot depicting PrimPol protein levels in eHAP iCas9 WT cells or PrimPol KO cells complemented with WT PrimPol, PrimPol AxA or PrimPol CH mutants. Exogenous protein levels were downregulated using the misFIT expression tuner system. h , Cell viability measured using the Cell Titer Glo assay on treatment of the indicated doses of cisplatin over 5 d. These experiments were performed in n = 4 biological replicates. Data are presented as mean ± s.d. i , Experimental scheme for a Cell Titer Glo assay to determine cell viability of eHAP iCas9 WT or PrimPol KO cells challenged with the indicated drugs. j , Cell viability measured after challenging eHAP iCas9 WT or PrimPol KO cells with the indicated doses of BPDE. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. k , Cell viability measured after challenging eHAP iCas9 WT or PrimPol KO cells with the indicated doses of MMC. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. l , A heatmap depicting sensitivity of eHAP iCas9 PrimPol KO versus WT cells to various DNA-damaging agents and inhibitors to DDR proteins. Sensitivities were determined by computing the log 2 (fold-change) between half-maximal inhibitory concentration (IC 50 ) values of PrimPol KO and WT cells. Source numerical data and unprocessed blots are available in the source data. ATMi, ataxia-telangiectasia mutated (ATM) inhibitor; ATRi, ATR inhibitor; FC, fold-change; HU, hydroxyurea; MMS, methyl methane sulfonate; Rad51i, Rad51 inhibitor; TS, template switching.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: CRISPR, Infection, Genome Wide, Western Blot, Viability Assay, Mutagenesis, Expressing, Glo Assay, Concentration Assay

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) DECODR analysis of Sanger sequencing of the PRIMPOL PR sgRNA cut site in eHAP iCas9 PRIMPOL KO C1 cells showing the cells habour a -2 bp deletion. ( b ) Experimental scheme for assessing inducible Cas9 activity using a lentiviral BFP/GFP flow cytometry reporter assay. ( c ) Representative gating strategy for all flow cytometry experiments conducted in this study. ( d ) Flow cytometry of eHAP iCas9 WT or PRIMPOL KO C1 cells infected with the BFP/GFP reporter shown in (B) in the presence or absence of doxycycline. ( e ) Sanger sequencing of the PRIMPOL AxA mutant (D114A/E116A) cDNA. ( f ) Sanger sequencing of the PRIMPOL CH mutant (C419H/H426Y) cDNA. ( g ) Scheme for introducing microRNA response elements (MREs) into the 3’ UTRs of PRIMPOL expression constructs to fine-tune protein expression levels. ( h ) A Western blot showing PRIMPOL expression levels in eHAP iCas9 WT (lane 1) or PRIMPOL KO cells transiently transfected with PRIMPOL expression constructs containing indicated MREs. ( i ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the PARP inhibitor Olaparib. ( j ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the DNA polymerase inhibitor aphidicolin. ( k ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the DNA polymerase alpha inhibitor ST1926. ( l ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of bleomycin. ( m ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the methyl methane sulfonate (MMS). ( n ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the RAD51 inhibitor B02. ( o ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the ribonucleotide reductase inhibitor hydroxyurea (HU). ( p ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the ATR inhibitor AZD6738. ( q ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the topoisomerase I inhibitor camptothecin (CPT). ( r ) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of ATM inhibitor KU-5593. All experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Sequencing, Activity Assay, Flow Cytometry, Reporter Assay, Infection, Mutagenesis, Expressing, Construct, Western Blot, Transfection

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , A volcano plot measuring statistical significance against log 2 (fold-change) in sgRNA counts in eHAP iCas9 PrimPol KO cells versus WT cells in the cisplatin-treated arm of the pool 1 sgRNA screen on day 6. Labelled genes exhibited increased sgRNA counts in PrimPol KO cells versus WT cells. b , A volcano plot measuring statistical significance against log 2 (fold-change) in sgRNA counts in eHAP iCas9 PrimPol KO cells versus WT cells in the cisplatin-treated arm of the pool 2 sgRNA screen on day 6. Genes labelled in blue exhibited increased sgRNA counts whereas genes labelled in red exhibited decreased sgRNA counts in PrimPol KO cells versus WT cells. c , A histogram of curated messenger RNA sequencing data depicting the expression of SLFN11 in human cancer cell lines curated from the DepMap repository. d , A western blot (WB) showing SLFN11 and PrimPol expression levels in HeLa, U2OS, HCT116, A549, HEK293A, DU145, eHAP and A673 cells infected with lentiviruses harbouring either Cas9–AAVS1 or Cas9–PrimPol. e , Experimental scheme showing how HeLa, U2OS, HCT116, A549, HEK293A, DU145, eHAP and A673 cells infected with Cas9–sgAAVS1 or Cas9–sgPrimPol were challenged with cisplatin and cell viability measured using the Cell Titer Glo assay. f – n , Cisplatin dose–response curves for HeLa Kyoto ( f ), U2OS ( g ), HCT116 ( h ), RPE-1 p53 KO ( i ), A549 ( j ), DU145 ( k ), HEK293A ( l ), A673 ( m ) and eHAP ( n ) cell lines harbouring Cas9–AAVS1 or Cas9–PrimPol. All experiments were performed in n = 2 biological replicates. Data are presented as means. Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: RNA Sequencing, Expressing, Western Blot, Infection, Glo Assay

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) An experimental scheme demonstrating how viability assays were performed in indicated PRIMPOL KO cell lines. ( b ) A Western blot depicting PRIMPOL and SLFN11 protein levels in indicated cell lines and PRIMPOL KO clones. ( c ) A cisplatin dose response curve for HT-1080 ICas9 WT or PRIMPOL KO cells. Experiments were performed in N = 2 biological replicates. Data are represented as means ± SD. ( d ) Cisplatin dose response curves for NCIH-460 iCas9 WT or PRIMPOL KO clones. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. ( e ) A cisplatin dose response curve for A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL KO clones. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Western Blot, Clone Assay

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , A schematic depicting how viability assays were performed. CTG, Cell Titer Glo. b , A western blot demonstrating transient knockout of SLFN11 in eHAP iCas9 WT or PrimPol KO cells. c , A dose–response curve of eHAP iCas9 WT or PrimPol KO cells in response to the indicated doses of cisplatin on loss of SLFN11. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. These experiments were performed using the same WT sgNTC and PrimPol KO sgNTC samples depicted in Fig. . d , An experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with cisplatin in the presence or absence of A-92, a chemical inhibitor of GCN2. e , A schematic depicting the signalling cascade connecting DNA damage to the ISR factor GCN2 and subsequent ribosome stalling and cell death. Inhibition of GCN2 prevents ribosome stalling and downstream cell death. f , Cisplatin dose–response curves for eHAP iCas9 WT or PrimPol KO cells in the presence or absence of 750 nM GCN2 inhibitor A-92. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. g , An experimental scheme for measuring apoptosis via cleaved caspase-3 using flow cytometry. h , A bar plot depicting the percentage of cleaved caspase-3 positive cells determined using flow cytometry as shown in g . Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to determine biological significance. P values: <0.0001 (WT versus PrimPol KO sgNTC + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). i , An experimental scheme for measuring cell viability in response to cisplatin. j , A western blot showing SLFN11, PrimPol and GFP protein expression in SLFN11 WT, SLFN11 E209A (tRNA nuclease mutant), SLFN11 Lys652Asp (ssDNA-binding mutant) or SLFN11 Glu669Gln (helicase mutant) re-expression cell lines. k , Dose–response curves for SLFN11 re-expression cell lines challenged with cisplatin. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. l , An experimental setup depicting how chromatin-fractionation immunofluorescence experiments were performed. m , The eHAP iCas9 WT or PrimPol KO cells transiently depleted of SLFN11 mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of phospho-RPA32 Ser33, DAPI or merged are shown. n , Quantification of sum focal intensity of phospho-RPA32 Ser33 in the indicated cell lines. Data were normalized in each biological replicate to untreated WT samples. These experiments were performed in n = 5 biological replicates. Data are presented as mean ± s.d. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.0004 (WT versus PrimPol KO sgNTC + cisplatin); 0.2141 (WT sgNTC versus PrimPol KO sgSLFN11 + cisplatin); 0.1980 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). The pRPA S33 staining was performed as a co-stain with γH2AX phospho-Ser139 depicted in Extended Data Fig. . o , A representative western blot depicting levels of RPA phospho-Ser33, SLFN11 and GCN2 phospho-Thr899 in whole-cell extracts and chromatin fractions after treatment with 1 μM cisplatin for 24 h. This blot was performed in n = 2 biological replicates. Source numerical data and unprocessed blots are available in the source data. s.e., short exposure; l.e., long exposure.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Western Blot, Knock-Out, Inhibition, Flow Cytometry, Expressing, Mutagenesis, Binding Assay, Fractionation, Immunofluorescence, Staining

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) A schematic depicting how viability assays were performed. ( b ) Representative images of colony formation assays used to assess viability of eHAP iCas9 WT or PRIMPOL KO cells in response to cisplatin upon loss of SLFN11. ( c ) Quantitation of colony formation assays as depicted in (b). These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was used to calculate biological significance. P -values: 0.0057 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin). ( d ) Experimental scheme for a lentiviral-based mCherry/GFP Cas9 flow cytometry reporter. ( e ) Flow cytometry of A673 Cas9-AAVS1 C2 or A673 Cas9-PRIMPOL C2 cells showing Cas9 activity in these cells. ( f ) Experimental scheme for challenging A673 Cas9-AAVS1 C2 or A673 Cas9-PRIMPOL C2 cells transiently depleted of SLFN11 protein with cisplatin. ( g ) A Western blot showing PRIMPOL and SLFN11 protein levels in A673 Cas9-AAVS1 C2 and A673 Cas9-PRIMPOL cells transiently depleted of SLFN11 protein. ( h ) Bar blots showing cell survival of A673 Cas9-AAVS1-C2 or A673 Cas9-PRIMPOL C2 cells transiently depleted of SLFN11 protein in the presence of 58 nM cisplatin. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A one-way ANOVA was performed to assess biological significance. P -values: 0.0418 (A673 Cas9-AAVS1 sgNTC vs. A673 Cas9-PRIMPOL sgNTC); 0.0214 (A673 Cas9-PRIMPOL sgNTC vs. A673-Cas9 PRIMPOL sgSLFN11). ( i ) Bar plots showing cell survival of eHAP iCas9 WT or PRIMPOL KO cells in response to treatment with 750 nM GCN2 inhibitor in the absence of cisplatin. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess biological significance. P -values: >0.9999 (WT vs. WT + GCN2i); 0.7360 (PRIMPOL KO vs. PRIMPOL + GCN2i). ( j ) Experimental scheme for challenging eHAP iCas9 WT or PRIMPOL KO cells with cisplatin in the presence or absence of GCN2 inhibitor following transient knockout of SLFN11. ( k ) A dose response curve depicting cells with indicated genotypes challenged with cisplatin in the absence (solid lines, circles) or presence (dashed lines, triangled) of 750 nM GCN2 inhibitor. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. ( l ) An experimental scheme for challenging eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, or RAD18 KO cells with cisplatin in the presence or absence of 750 nM GCN2 inhibitor. ( m ) A Western blot showing PRIMPOL, FANCD2, and RAD18 protein levels in eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, and RAD18 KO cells. ( n ) A bar plot depicting cell survival of eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, and RAD18 KO cells at 450 nM cisplatin compared to an untreated control in each cell line in the absence (darker bars) or presence (lighter bars) of GCN2 inhibitor. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: 0.9939 (WT vs. WT + GCN2i); 0.0006 (PRIMPOL KO vs. PRIMPOL KO + GCN2i); 0.9901 (FANCD2 KO vs. FANCD2 KO + GCN2i); 0.2275 (RAD18 KO vs. RAD18 KO + GCN2i); 0.9998 (WT + GCN2i vs. PRIMPOL KO + GCN2i); <0.0001 (WT + GCN2i vs. FANCD2 KO + GCN2i); <0.0001 (WT + GCN2i vs. RAD18 KO + GCN2i). ( o ) An experimental scheme for challenging eHAP iCas9 WT cells with cisplatin in the presence or absence of RAD51 inhibitor (B02) following transient knockout of SLFN11. ( p ) A bar plot depicting cell survival in untreated conditions in the absence or presence of RAD51 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: >0.9999 (WT sgNTC vs. WT sgSLFN11); >0.9999 (WT sgNTC + 4.5 µM RAD51i vs. WT sgSLFN11 + 4.5 µM RAD51i); 0.9858 (WT sgNTC 9.0 µM RAD51i vs. WT sgSLFN11 + 9.0 µM RAD51i). ( q ) A bar plot depicting cell survival in cells treated with 450 nM cisplatin in the absence or presence of RAD51 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: >0.9999 (WT sgNTC vs. WT sgSLFN11); >0.9999 (WT sgNTC + 4.5 µM RAD51i vs. WT sgSLFN11 + 4.5 µM RAD51i); 0.9982 (WT sgNTC 9.0 µM RAD51i vs. WT sgSLFN11 + 9.0 µM RAD51i). Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Quantitation Assay, Flow Cytometry, Activity Assay, Western Blot, Knock-Out, Control

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , A schematic depicting how cell viability assays were performed in eHAP iCas9 WT or PrimPol KO cells challenged with cisplatin after transient knockout of USP1 or WDR48. b , A representative western blot depicting PrimPol, USP1 and WDR48 protein levels on loss of USP1 or WDR48. This western blot was performed in n = 2 biological replicates. c , A cisplatin dose–response curve for eHAP iCas9 WT or PrimPol cells on loss of USP1. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. d , A cisplatin dose–response curve for eHAP iCas9 WT or PrimPol cells on loss of WDR48. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. These experiments were performed using the same WT sgNTC and PrimPol KO sgNTC samples depicted in Fig. . e , An experimental scheme for measuring RPA exhaustion in cell pools after transient knockout of USP1 and SLFN11 using QIBC. f , A representative western blot showing USP1 and SLFN11 protein levels after transient knockout of each protein. This western blot was performed in n = 2 biological replicates. g , Representative scatter plots depicting mean ssDNA (BrdU) intensity ( y axis) plotted against mean chromatin-bound RPA32 intensity ( x axis) for each genotype and treatment depicted. The dashed boxes indicate RPA-exhausted cells where the ratio of mean ssDNA signal against mean chromatin-bound RPA32 signal has deviated from linearity (blue line). The percentage of cells undergoing RPA exhaustion in each genotype and treatment is shown. h , A bar plot depicting the percentage of cells undergoing RPA exhaustion in each genotype and treatment tested. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: <0.0001 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and <0.0001 (PrimPol KO sgSLFN11 versus PrimPol KO sgSLFN11–USP1 + cisplatin). i , An experimental scheme depicting how apoptosis (cleaved caspase-3 signal) was measured using flow cytometry in eHAP iCas9 cells after transient knockout of USP1 or SLFN11. j , A bar plot depicting the percentage of cells undergoing apoptosis, as assessed by measuring the cleaved caspase-3 signal using flow cytometry. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgUSP1 + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgUSP1–SLFN11 + cisplatin); 0.0038 (PrimPol KO sgUSP1 versus PrimPol KO sgSLFN11 + cisplatin); and >0.9999 (PrimPol KO sgSLFN11 versus PrimPol KO sgSLFN11–USP1 + cisplatin). Source numerical data and unprocessed blots are available in the source data. DOX, doxycycline.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Knock-Out, Western Blot, Flow Cytometry

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) Representative flow cytometry experiments depicting cleaved caspase-3 signal plotted against forward scatter area. Cleaved caspase-3 positive cells were gated and quantitated as depicted. ( b ) A scheme depicting how apoptosis was measured in eHAP iCas9 WT or PRIMPOL KO cells using cleaved caspase-3 positive cells in flow cyomtetry experiments. ( c ) Representative dot plots depicting how the percentage of cleaved caspase-3 positive cells were determined using flow cytometry after challenge with cisplatin in the absence or presence of 650 nM GCN2 inhibitor. ( d ) A bar plot depicting the percentage of cleaved caspase-3 positive cells in each indicated genotype following challenge with cisplatin in the absence or presence of GCN2 inhibitor determined using flow cytometry in (b). These experiments were performed in N = 4 biological replicates. Data represented as means ± SD. A two-way ANOVA was performed to assess biological significance P -values: 0.0003 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); >0.9999 (WT sgSLFN11 vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.0002 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). ( e ) Representative Western blots demonstrating induction of DNA damage response markers in eHAP iCas9 WT or PRIMPOL KO cells in response to indicated doses of cisplatin for 24 or 48 h. ( f ) Experimental setup of measuring chromatin-bound phospho-gamma H2AX serine 139 using immunofluorescence in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein (top). ( g ) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of γH2AX phospho serine 139, DAPI, or merged are shown. ( h ) Bar plots showing phospho-gamma H2AX serine 139 foci signal normalised to WT untreated cells in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein. Experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was used to assess biological significance. P -values: >0.9999 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.4353 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.2441 (WT sgNTC vs. PRIMPOL KO sgSLFN11). γH2AX phospho serine 139 staining was performed as a co-stain with pRPA S33 depicted in Fig. . ( i ) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of RAD51, DAPI, or merged are shown ( j ) Quantification of RAD51 foci in indicated cell lines. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess biological significance. P -values: 0.0050 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.0013 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.9994 (WT sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Flow Cytometry, Western Blot, Immunofluorescence, Staining

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , A diagram depicting how ssDNA was measured by detecting incorporated BrdU under non-denaturing conditions. b , An experimental scheme for measuring RPA exhaustion in eHAP iCas9 WT or PrimPol KO cells in which SLFN11 has been transiently knocked out using QIBC. c , Representative scatter plots depicting mean BrdU signal intensity per cell ( y axis) plotted against mean chromatin-bound RPA32 ( x axis). The linear relationship between BrdU and RPA32 signals is depicted as a light-blue line. Cells undergoing RPA exhaustion are depicted within the dashed box and coloured red. The percentage of cells undergoing RPA exhaustion is indicated in each panel. d , A bar plot depicting the percentage of cells undergoing RPA exhaustion in the experiments shown in a and b . These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: 0.0009 (WT sgNTC versus PrimPol KO sgNTC + cisplatin); <0.0001 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and 0.0009 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). e , Representative scatter plots depicting mean γH2AX–pSer139 signal intensity per cell ( y axis) plotted against mean chromatin-bound RPA32 ( x axis). Cells with high RPA32 and γH2AX–pSer139 signals are coloured in red. The percentage of cells with high RPA32 and γH2AX–pSer139 signals is indicated in each panel. f , A bar plot depicting the percentage of cells exhibiting high RPA32–γH2AX–pSer139 signals in the experiments shown in a and e . These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: 0.0023 (WT sgNTC versus PrimPol KO sgNTC + cisplatin); 0.0003 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and 0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). g , Experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with cisplatin in the presence or absence of GCN2i after siRNA knockdown of RPA2. h , A western blot depicting RPA2, PrimPol, vinculin and total protein levels in eHAP iCas9 WT or PrimPol KO cells after transient knockdown of RPA2. i , A dose–response curve depicting cell survival in eHAP iCas9 WT or PrimPol KO cells (normalized to untransfected cells) after siRNA knockdown of RPA at indicated concentrations in the presence or absence of cisplatin and GCN2i. This experiment was performed in n = 3 biological replicates. Data are presented mean ± s.d. j , An experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with two fixed doses of cisplatin in the presence or absence of DNA-PK inhibitor (DNA-PKi) after transient knockout of SLFN11. k , A bar plot depicting cell survival in the presence of 450 nM cisplatin with or without treatment with DNA-PKi. The experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess statistical significance. P value: <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). l , A bar plot depicting cell survival in the presence of 1.5 μM cisplatin with or without treatment with DNA-PKi. The experiments shown were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess statistical significance. P values: 0.0423 (WT sgNTC − DNA-PKi versus WT sgNTC + DNA-PKi); <0.0001 (WT sgSLFN11 − DNA-PKi versus WT sgSLFN11 + DNA-PKi); <0.0001 (PrimPol KO sgSLFN11 − DNA-PKi versus PrimPol sgSLFN11 + DNA-PKi).

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Knockdown, Western Blot, Knock-Out

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) Representative micrographs depicting BrdU, RPA, and DAPI signals generated in QIBC experiments shown in Fig. . ( b ) Representative micrographs depicting γH2AX pS139, RPA, and DAPI signals generated in QIBC experiments shown in Fig. . ( c ) A dose response curve depicting cell viability in eHAP iCas9 WT or PRIMPOL KO cells treated with indicated doses of siRNA targeting the RPA2 gene in the presence or absence of 750 nM GCN2 inhibitor. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Generated

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) An experimental schematic describing how cell viability assays were performed in the presence of cisplatin and ML323 (USP1 inhibitor). ( b ) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells with or without 4.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. ( c ) A bar plot showing how eHAP iCas9 WT (gray bars) or PRIMPOL KO cells (red bars) respond to 4.5 μM ML323. Survival is normalised to untreated conditions within each genotype. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: 0.4946 (WT vs. WT + USP1i); 0.7018 (PRIMPOL KO vs. PRIMPOL KO + USP1i). ( d ) A schematic showing how cell viability experiments were performed in A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of cisplatin and ML323 (16.5 μM). ( e ) A bar blot depicting cell survival for A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of 200 nM cisplatin -/+ 16.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: 0.9925 (WT vs. WT + USP1i); 0.0203 (PRIMPOL KO vs. PRIMPOL KO + USP1i). ( f ) An experimental scheme depicting how cell viability was measured after challenging cells with cisplatin and USP1 inhibitor (ML323) in eHAP iCas9 cells with indicated genotypes. ( g ) A bar plot depicting cell survival of eHAP iCas9 WT or PRIMPOL KO cells following challenge with 670 nM cisplatin in the absence (darker bars) or presence (light bars) of 4.5 μM USP1 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P -values: >0.9999 (WT sgNTC vs. WT sgNTC + USP1i); >0.9999 (WT sgSLFN11 vs. WT sgSLFN11 + USP1i); 0.0032 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgNTC + USP1i); 0.8134 (PRIMPOL KO sgSLFN11 vs. PRIMPOL KO sgSLFN11 + USP1i); 0.8522 (WT sgNTC vs. WT sgSLFN11); <0.0001 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11); 0.8160 (WT sgNTC + USP1i vs. PRIMPOL KO sgNTC + USP1i). Source numerical data are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Knock-Out

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: ( a ) A schematic showing how RAD18 or FANCL was knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin. ( b ) A Western blot showing PRIMPOL and RAD18 protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of RAD18 (R18). ( c ) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL cells upon loss of RAD18. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. These experiments were performed using the same WT sgNTC and PRIMPOL KO sgNTC samples depicted in (m). ( d ) A Western blot showing PRIMPOL and FANCL protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL (FL). ( e ) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL. eHAP iCas9 WT and PRIMPOL KO curves are the same as those shown in (d), as these experiments were performed at the same time. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. ( f ) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL cells upon loss of USP1 and/or RAD18 (R18). ( g ) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of USP1 and/or FANCL (FL). ( h ) An experimental scheme for how USP1/RAD18/FANCL proteins were knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin in a colony formation assay. ( i ) Representative images of colony formation assays in indicated cell backgrounds in untreated conditions or upon treatment with 450 nM cisplatin. ( j ) Quantification of colony survival in the colony formation assays represented in (d) normalised to untreated samples within each genotype. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to test statistical significance. P -values: 0.9964 (WT sgNTC vs. WT sgUSP1); 0.0098 (WT sgRAD18/NTC vs. WT sgRAD18/sgUSP1); 0.9998 (WT sgFANCL/sgNTC vs. WT sgFANCL/USP1); 0.0012 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgUSP1); <0.0001 (PRIMPOL KO sgRAD18/NTC vs. PRIMPOL KO sgRAD18/USP1); >0.9999 (PRIMPOL KO sgFANCL/NTC vs. PRIMPOL KO sgFANCL/USP1). ( k ) An experimental schematic describing how chromatin-bound immunofluorescence was performed. ( l ) Representative micrographs depicting DNA staining (DAPI), FANCD2 staining, and a merged image of DAPI/FANCD2 staining. ( m ) A bar plot showing chromatin-bound FANCD2 foci numbers in eHAP iCas9 WT or PRIMPOL KO cells treated with 450 nM cisplatin. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.0071 (WT + cisplatin vs. PRIMPOL KO + cisplatin). Source numerical data and unprocessed blots are available in the source data.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Western Blot, Colony Assay, Immunofluorescence, Staining

Journal: Nature Cell Biology

Article Title: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress

doi: 10.1038/s41556-025-01852-1

Figure Lengend Snippet: a , An experimental scheme depicting how cell viability was measured in indicated cell lines after transient knockout of SLFN11 and challenge with DNA polymerase α inhibitor (ST1926). b , A western blot depicting SLFN11 protein levels after transient knockout of SLFN11 in the indicated cell lines. c – g , Dose–response curves of HeLa Kyoto iCas9 ( c ), HT-1080 iCas9 ( d ), TOV112D iCas9 ( e ), A673 iCas9 ( f ) and eHAP iCas9 ( g ) cells challenged with the indicated doses of DNA polymerase α inhibitor (ST1926). Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. h , In WT cells expressing SLFN11 (blue panel), PrimPol repriming activity restricts accumulation of ssDNA at cisplatin-stalled replication forks caused by uncoupling of the helicase and DNA polymerase, thereby preventing SLFN11 activation and cell death. Similarly, inhibition of DNA polymerase α uncouples DNA replication on each strand to drive ssDNA accumulation at forks in a PrimPol-independent manner. In PrimPol-deficient cells (green panel), cisplatin-induced DNA damage drives accumulation of ssDNA at replication forks in a USP1-dependent manner, leading to RPA exhaustion and SLFN11 activation. USP1-dependent downregulation of the FA pathway allows for full SLFN11 activation and subsequent ribosome stalling, ISR activation and cell death, as previously described . In SLFN11-deficient cells (red panel), cisplatin-induced DNA damage or inhibition of DNA polymerase α causes ssDNA accumulation at forks and RPA exhaustion. Loss of SLFN11 prevents signal transduction through ribosome stalling and GCN2, preventing SLFN11-dependent cell death. Instead, prolonged RPA exhaustion leads to fork breakage and replication catastrophe as previously described , leading to DSBs that are repaired by the NHEJ pathway, leading to cell death. Source numerical data and unprocessed blots are available in the source data. PolAi, Pol A inhibitor.

Article Snippet: PrimPol cDNA was purchased from OriGene (catalogue number SC100629).

Techniques: Knock-Out, Western Blot, Expressing, Activity Assay, Activation Assay, Inhibition, Transduction

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Schematic showing the sequence of PrimPol's RBM-A (residues 510–528), located in the C-terminal RBD (residues 480–560). ( b ) 15 N- 1 H HSQC spectra showing RPA70N in the absence (black) or presence (red) of twofold molar excess of unlabelled RBM-A peptide. ( c ) Electrostatic surface model of RPA70N with RBM-A (green) bound in the basic cleft. Basic and acidic surfaces are coloured blue and red, respectively. ( d ) Key stabilizing interactions of RBM-A (green) in the RPA70N basic cleft (purple). RBM-A binds between β sheets in the β barrel of RPA70N. Of particular importance for binding are the electrostatic interactions of D519 with the side chains of two arginines (R31 and R43) in the RPA70N basic cleft.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) The sequence of PrimPol's RBM-B (residues 542–560), located in the C-terminal RBD (residues 480–560). ( b ) The continuous electron density of RBM-B residues 548–556 in the complex with RPA70N. ( c ) Electrostatic surface model of RPA70N with RBM-B (green) bound in the basic cleft. Basic and acidic surfaces are coloured blue and red, respectively. ( d ) 15 N- 1 H HSQC spectra showing RPA70N in the absence (black) or presence (red) of a twofold molar excess of unlabelled RBM-B peptide. ( e ) Key stabilizing interactions of RBM-B (green) in the RPA70N basic cleft (purple). RBM-B binds between β sheets in the β barrel of RPA70N. D551 is of particular importance as it forms a number of electrostatic interactions with both the side chains and a backbone amide NH of the RPA70N peptide.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Sequencing

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Mutation of RBM-A does not abolish binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD A-KO (purple), RPA70N (black) and RBD A-KO with RPA70N in a 1:1 ratio (green). ( b ) 15 N- 1 H HSQC spectra showing RPA70N alone (black), in the presence of twofold molar excess of either RBD A-KO (green) or RBM-B peptide (residues 542–560) (red). The perturbations observed for RBD A-KO are similar to those induced by the RBM-B peptide. ( c ) Truncation of RBM-B does not prevent binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD B-KO (purple), RPA70N (black) and RBD B-KO with RPA70N in a 1:1 ratio (blue). ( d ) 15 N- 1 H HSQC spectra showing RPA70N alone (black) or in the presence of twofold molar excess of RBD B-KO (blue) or RBM-A peptide (residues 510–528) (red). The perturbations observed for RBD B-KO are similar to those induced by the RBM-A peptide. ( e ) Mutation of both RBM-A and RBM-B abolishes the binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD A/B-KO (purple), RPA70N (black) and RBD A/B-KO with RPA70N in a 1:1 ratio (red). ( f ) 15 N- 1 H HSQC spectra showing RPA70N alone (black) or in the presence of twofold molar excess of RBD A/B-KO (red). The near identity of the two spectra indicates there is no interaction.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Schematic detailing the domain architecture of N-terminal FLAG-tagged PrimPol transfected into HEK-293 derivative cells (Flp-In T-Rex-293). The RBD (480–560) containing the RBM-A and B sites is shown below with the mutations forming the A-KO (D519R and F522A) and B-KO (D551R and I554A) highlighted. ( b ) Flp-In T-Rex-293 cells were transfected with wild-type and RBM-A and B mutated PrimPol. Expression was confirmed by addition of 10 ng ml −1 doxycycline (indicated by Dox ± on figure) for 24 h and subsequent western blotting. ( c ) Flp-In T-Rex-293 cells transfected with FLAG-tagged wild-type (WT) PrimPol were grown in the presence or absence of doxycycline (10 ng ml −1 , 24 h), FLAG-PrimPol was immunoprecipitated from the soluble cell lysate using anti-FLAG antibody and western blotted for PrimPol (anti-FLAG) and RPA (anti-RPA2). The presence and absence of doxycycline is indicated by ± Dox, ‘In' indicates the input, ‘E1', ‘E2' and ‘E3', indicate elutions 1, 2 and 3, respectively. ( d ) Immunoprecipitation of FLAG-PrimPol A-KO (D519R/F522A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. ( e ) Immunoprecipitation of FLAG-PrimPol B-KO (D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. ( f ) Immunoprecipitation of FLAG-PrimPol A+B-KO (D519R/F522A and D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) PrimPol −/− DT40 cells were complemented with un-tagged human PrimPol constructs; wild-type hPrimPol (+ WT), hPrimPol D519R/F522A (+ A-KO) and hPrimPol D551R/I554A (+ B-KO). ‘WT' indicates lysate from wild-type DT40 cells, ‘−/−‘ indicates lysate from PrimPol −/− DT40 cells. ( b ) DNA fibre analysis was performed on DT40 cells expressing each PrimPol construct. Cells were ultraviolet-C irradiated (20 J m −2 ) between the CldU and IdU labelling periods (each 20 min). Representative DNA fibres showing 1:1, 2:1 and 3:1 CldU:IdU ratios are presented; >100 individual DNA fibres were scored for each experiment. ( c ) Mutation of RBM-A causes increased fork stalling following ultraviolet-C irradiation. Data are representative of the means of three individual experiments and were subject to an unpaired t -test showing a significant difference between the mean CldU/IdU ratio for the ‘+ WT hPrimPol' and ‘+ A-KO hPrimPol' data sets ( P <0.05). ( d ) DNA fibre analysis from the ‘+ A-KO hPrimPol' DT40 cells presented as a cumulative percentage of forks at each ratio. ( e ) Mutation of RBM-B does not significantly alter the level of fork stalling following ultraviolet-C irradiation. DNA fibre analysis of the ‘+ B-KO hPrimPol' DT40 cells, showing the percentage of forks at each CldU:IdU ratio. Data are representative of the means of three individual experiments. ( f ) DNA fibre analysis from the ‘+ B-KO hPrimPol' DT40 cells presented as a cumulative percentage of forks at each ratio.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Construct, Expressing, Irradiation, Mutagenesis

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) PrimPol's RBM-A, but not RBM-B, is critical for recruitment to chromatin. Flp-In T-Rex-293 cells transfected with WT and RBM-A and B mutant PrimPol constructs were either mock (−) or ultraviolet-C (30 J m −2 ) (+) irradiated before separation into Triton X-100 (0.5%) soluble and insoluble fractions. Samples were analysed by western blot alongside whole-cell extracts. Only insoluble samples are presented here, whole-cell extracts and soluble blots can be found in . ( b ) PrimPol's RBD is recruited to Xenopus egg extract chromatin in response to aphidicolin treatment, RBM-A is critical for this recruitment. Recombinant hPrimPol GST constructs (4 ng μl −1 ) were added to Xenopus egg extract supplemented with sperm nuclei (3 × 10 3 μl −1 ). Extract was treated with aphidicolin 100 μg ml −1 and incubated at 21 °C for 80 min. Chromatin was isolated and associated proteins analysed by SDS–PAGE and western blotting using the antibodies indicated. ( c ) Low concentrations of RPA stimulate PrimPol's primase activity, high concentrations inhibit. Primer synthesis by WT hPrimPol (400 nM) on M13 ssDNA templates (20 ng μl −1 ) in the presence of increasing concentrations of RPA. ‘C' indicates the no enzyme control, oligonucleotide (Nt) length markers are shown on the left of the gel. ( d ) Quantification of data shown in ‘c'. For each RPA concentration the fold increase in primer synthesis relative to reactions containing no RPA was calculated. Data are representative of three repeat experiments. ( e ) Schematic showing the effect of increasing RPA concentrations on PrimPol's primase activity. When no RPA is present a proportion of PrimPol binds to the M13 template and facilitates primer synthesis (left). When low/moderate concentrations of RPA are present PrimPol is recruited to the RPA/ssDNA interface causing an increase in primer synthesis activity (middle). At high RPA concentrations the M13 DNA template is fully saturated, blocking access of PrimPol to the DNA and inhibiting primer synthesis (right).

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Transfection, Mutagenesis, Construct, Irradiation, Western Blot, Recombinant, Incubation, Isolation, SDS Page, Activity Assay, Control, Concentration Assay, Blocking Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Unrepaired DNA damage lesions, or DNA secondary structures, in the leading strand template lead to stalling of polymerase ɛ. This causes uncoupling of replication, generating ssDNA downstream of the DNA damage lesion/structure and facilitating binding of RPA. Note that for simplicity other replisome components and lagging strand synthesis machinery are not shown. ( b ) PrimPol is recruited to the ssDNA interface uncovered by the replicative helicase through the interaction of its RBM-A with RPA70N. This interaction is stabilized by the binding of the ZnF and AEP domains to ssDNA. ( c ) PrimPol catalyses the synthesis of a new DNA primer, before further extension is prevented by the restraining effect of the RPA interaction and ZnF domain, coupled with the enzyme's low processivity. ( d ) Unable to continue with primer extension, PrimPol dissociates from the template strand. Re-binding upstream is prevented by RPA. ( e ) The nascent primer is utilized by the replicative polymerase for continued DNA replication. This leaves behind a short RPA-coated ssDNA region opposite the lesion to be filled in by template switching or TLS.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Binding Assay