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 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