ps33 rpa2  (Bethyl)

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF depletion affects DNA replication dynamics and induces replication stress. (A, B) DNA fiber analysis examining the effect of NSMF on replication dynamics in HCT116 cells. For normal conditions, cells were sequentially labeled with CldU and IdU for 30 min each ( A ). For replication stress conditions, cells were treated with 2 mM hydroxyurea (HU) for 2 h between CldU and IdU pulses ( B ). Representative DNA fiber images (upper) and quantification of IdU tract lengths (lower). The median value is indicated, derived from the analysis of 200 or more IdU and CldU tracts per experimental condition. **** P < .0001, two-tailed Mann–Whitney test. ( C, D ) Quantification of newly fired origins ( C ), IdU-only fibers) and stalled forks ( D ), CldU-only fibers) in HCT116 cells transfected with siCtrl or siNSMF#1. A total of 300–350 fibers from 7 to 18 randomly selected non-overlapping images per condition were analyzed. Data represent mean ± SEM. * P < .05, **** P < .0001. Statistical significance was assessed using a Mann–Whitney test. Results are representative of three independent experiments. ( E ) BrdU pulse-chase analysis of cell-cycle kinetics in HCT116 cells transfected with siCtrl or siNSMF#1 following release from a HU block (2 mM, 12 h). Representative results from three independent experiments are shown. ( F ) Immunofluorescence analysis of phospho-RPA2 foci in control (shCtrl) and NSMF-depleted (shNSMF#1 and #2) HCT116 cells under normal (NT) or HU treatment (2 mM, 16 h) conditions. The number of phospho-RPA2 foci per cell was quantified from at least 54 cells across two independent experiments. Scale bar, 10 μm. Data are presented as median. * P < .05, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( G ) γH2AX staining for control (shCtrl) or NSMF-depleted (shNSMF#2) HCT116 cells treated with HU treatment (2 mM, 16 h), and directly fixed or allowed to grow in the complete medium for 5 h in the absence of HU. The number of γH2AX foci per cell was quantified from at least 106 cells across three independent experiments. Scale bar, 10 μm. Data are presented as median. **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: The following primary antibodies were used for western blot Antibodies against NSMF (Aviva System Biology, Cat# OAAN03468), γH2AX (Cell Signaling Technology, Cat# 9718), β-Actin (GeneTex, Cat# GTX629630), GAPDH (Santa Cruz, Cat# sc-32233), α-Tubulin (GeneTex, Cat# GTX112141), H2B (Millipore, Cat# 07-371), pT1989-ATR (Cell Signaling Technology, Cat# 30632), ATR (Santa Cruz, Cat# sc-515173), pS345-CHK1 (Cell Signaling Technology, Cat# 2348), pS317-CHK1 (Cell Signaling Technology, Cat# 2344), CHK1 (Santa Cruz, Cat# sc-8408), pS33-RPA2 (Bethyl Laboratories Inc., Cat# A300-246A), RPA2 (Bethyl Laboratories Inc., Cat# A300-244A), p16^INK4A (Cell Signaling Technology, Cat# 80 772), p21 Waf1/Cip1 (Cell Signaling Technology, Cat# 2947), Ras (Cell Signaling Technology, Cat# 14412), Cyclin D1 (Cell Signaling Technology, Cat# 2978), PARP (Cell Signaling Technology, Cat# 9532), Cleaved PARP (Cell Signaling Technology, Cat# 9541), Caspase 3 (Cell Signaling Technology, Cat# 9662), and Cleaved Caspase 3 (Cell Signaling Technology, Cat# 9661).

Techniques: Labeling, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Pulse Chase, Blocking Assay, Immunofluorescence, Control, Staining

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF depletion affects DNA replication dynamics and induces replication stress. (A, B) DNA fiber analysis examining the effect of NSMF on replication dynamics in HCT116 cells. For normal conditions, cells were sequentially labeled with CldU and IdU for 30 min each ( A ). For replication stress conditions, cells were treated with 2 mM hydroxyurea (HU) for 2 h between CldU and IdU pulses ( B ). Representative DNA fiber images (upper) and quantification of IdU tract lengths (lower). The median value is indicated, derived from the analysis of 200 or more IdU and CldU tracts per experimental condition. **** P < .0001, two-tailed Mann–Whitney test. ( C, D ) Quantification of newly fired origins ( C ), IdU-only fibers) and stalled forks ( D ), CldU-only fibers) in HCT116 cells transfected with siCtrl or siNSMF#1. A total of 300–350 fibers from 7 to 18 randomly selected non-overlapping images per condition were analyzed. Data represent mean ± SEM. * P < .05, **** P < .0001. Statistical significance was assessed using a Mann–Whitney test. Results are representative of three independent experiments. ( E ) BrdU pulse-chase analysis of cell-cycle kinetics in HCT116 cells transfected with siCtrl or siNSMF#1 following release from a HU block (2 mM, 12 h). Representative results from three independent experiments are shown. ( F ) Immunofluorescence analysis of phospho-RPA2 foci in control (shCtrl) and NSMF-depleted (shNSMF#1 and #2) HCT116 cells under normal (NT) or HU treatment (2 mM, 16 h) conditions. The number of phospho-RPA2 foci per cell was quantified from at least 54 cells across two independent experiments. Scale bar, 10 μm. Data are presented as median. * P < .05, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( G ) γH2AX staining for control (shCtrl) or NSMF-depleted (shNSMF#2) HCT116 cells treated with HU treatment (2 mM, 16 h), and directly fixed or allowed to grow in the complete medium for 5 h in the absence of HU. The number of γH2AX foci per cell was quantified from at least 106 cells across three independent experiments. Scale bar, 10 μm. Data are presented as median. **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: Antibodies against pS33-RPA2 (Bethyl Laboratories Inc., Cat# A300-246A) and γH2AX (Sigma–Aldrich, Cat# 05-636) were used for immunofluorescence analysis.

Techniques: Labeling, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Pulse Chase, Blocking Assay, Immunofluorescence, Control, Staining

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a A representative structural model of human RPA was generated using AlphaFold and aligned to the trimerization core observed in the crystal structure (PDB:1L1O). The RPA70, RPA32, and RPA14 subunits form a heterotrimer and harbor multiple oligosaccharide/oligonucleotide binding (OB) domains. A, B, C, and D are DNA-binding domains (DBDs). OB-F and the wh-domain are two protein-interaction domains. The cell cycle-specific sites of phosphorylation on RPA32 and RPA70 are denoted in red/yellow. b Mass spectrum of the RPA70 tryptic peptide showing phosphorylation at the T191 residue. MS analysis was performed using endogenous RPA70 immunoprecipitated from HCT116 cells arrested in mitosis using 50 ng/mL nocodazole for 18 h. c Sequence alignment of RPA70 (RFA1) reveals a conserved SP/TP motif. Asterisks (*) indicate positions which have a single, fully conserved residue, colon (:) indicates conservation between groups of strongly similar properties and period (.) indicates conservation between groups of weakly similar properties. d Mass spectrum of the RPA70 tryptic peptide showing phosphorylation at the T191 residue. Reactions extracted from in vitro kinase assay of recombinant human RPA incubated with CDK1/Cyclin B complex followed by MS-MS analysis.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Generated, Binding Assay, Phospho-proteomics, Residue, Immunoprecipitation, Sequencing, In Vitro, Kinase Assay, Recombinant, Incubation, Tandem Mass Spectroscopy

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Cartoon depicts the sites that are hyperphosphorylated at the N-terminus of RPA32 in response to DNA damage. It is unclear as to how disruption of RPA70-T191 phosphorylation affects the hyperphosphorylation of RPA32. CDK1-primed sites (phospho-S23 and S29) on RPA32 are indicated in blue. b Caspase 3/7 activity in cells treated with 100 ng/mL SN-38 for 21 h. Data corrected for background absorbance and plotted as fold change over vehicle (veh) control (0.1% DMSO). Error bars denote SEM. Mean of three independent experiments plotted. 2 to 3 wells were assayed per experiment. ** p = 0.0023 (unpaired two-tailed t test). c Cells treated briefly with 20 ng/mL SN-38 for 90 min or vehicle (0.1% DMSO). Blots probed with the indicated antibodies with Tubulin and Vinculin as loading controls. Blots are representative of three independent experiments. Arrow indicates hyperphosphorylated form of RPA32. d Cells synchronized with double thymidine block were released, and 7 hrs post-release (cells in G2) were treated with 100 μM etoposide for 45 min. Tubulin probed as a loading control. Blots are representative of three independent experiments. e Western blot analysis of cells treated with 500 μM MMS for 24 h. Blots probed with indicated antibodies with Tubulin as loading control. Blots are representative of three independent experiments. f For recovery, cells treated with 0.1% DMSO (vehicle) or 50 μM etoposide for 2 h, washed and allowed to recover for 22 h. For caspase 3/7 activity, data were corrected for background absorbance and expressed as fold over WT-vehicle. Error bars denote SEM. Mean of three independent experiments shown. 3 wells assayed per experiment. ** p = 0.0016 (One-way ANOVA- Sidak’s multiple comparison test). All molecular weight markers in the figure denoted in kDa.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Disruption, Phospho-proteomics, Activity Assay, Control, Two Tailed Test, Blocking Assay, Western Blot, Comparison, Molecular Weight

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Structural model of the domains of RPA modeled generated using AlphaFold. The positions of phosphorylation (T191 and S384 in RPA70 and S23 and S29 in RPA32 are depicted. b Circular dichroism (CD) spectra of RPA, RPA-T191A, and T191D does not show significant differences in the overall secondary structures. The ssDNA binding activity of RPA, RPA-T191A, and RPA-T191D were measured using fluorescence anisotropy and fluorescein-labeled ( c ) (dT) 35 or ( d ) (dT) 20 oligonucleotides. The ssDNA binding of all three RPA proteins are stoichiometric due to the high-affinity ssDNA binding activity of RPA. Stopped flow analysis of RPA-ssDNA interactions were performed by following the change in intrinsic Trp fluorescence changes as a function of increasing (dT) 35 concentrations. Data for ( e ) RPA, ( f ) RPA-T191A, and ( g ) RPA-T191D are shown. Data were fit to single exponentials and ( h ) the k obs values plotted again ssDNA concentration yields similar k ON values for ssDNA binding for all three RPA proteins (RPA = 1.08 ± 0.2 x 10 8 M −1 s −1 , RPA-T191A = 0.86 ± 0.3 x 10 8 M −1 s −1 , RPA-T191D = 1.06 ± 0.2 x 10 8 M −1 s −1 ). i–l Mass photometry analysis of RPA or RPA-T191D complexes on (dT) 50 ssDNA were performed at either 1:1 or 4:1 molar ratios of RPA:DNA. Predominantly single RPA-bound ssDNA complexes are observed at 1:1 ratios, and both 1:1 and 2:1 RPA-bound complexes are observed. However, there are no quantifiable differences in the ssDNA binding properties between RPA and RPA-T191D. m Size exclusion chromatography analysis of RPA, RPA-T191A, and RPA-T191D in the absence of presence of longer (dT) 97 ssDNA substrates shows similar profiles for RPA interactions. In these experiments, a three-fold excess of RPA was used to drive the assembly of multiple RPA molecules on the DNA. For all these experiments, representative data from a minimum of three independent experiments are shown. Error bars, where appropriate, are shown and represent St. Dev. values from n ≥ 3 independent experiments.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Generated, Phospho-proteomics, Circular Dichroism, Binding Assay, Activity Assay, Fluorescence, Labeling, Concentration Assay, Size-exclusion Chromatography

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Intrinsic Trp fluorescence scan of RPA versus RPA-T191D shows reduced signal for the phosphomimetic mutant. b Crosslinking mass spectrometry analysis of RPA with BS3. Crosslinks (XLs) within each subunit and between the three subunits are observed. The individual OB domains and the wh domain are denoted. The disordered N-terminus of RPA32 and the two protein interaction domains (F and wh) are highlighted by the dashed lines. c XL-MS analysis of RPA-T191D shows lesser overall XLs compared to RPA in panel ( b ). In particular, XLs originating from the two protein interaction domains (F and wh) and the N-terminus of RPA32 are all reduced. XLs in RPA14 are also reduced. Data suggest an overall configurational opening of RPA-T191D. d XL-MS analysis of the RPA T191D,S23D,S29D (RPA 3D ) triple phosphomimetic mutant shows patterns of crosslinking that are different compared to RPA or RPA-T191D. No XLs are captured in the N-terminus of RPA32, suggesting that this region is now fully accessible (dashed square). e Intrinsic Trp fluorescence scan of RPA 3D shows differences that are marginally higher than RPA. This observation is different than the intrinsic Trp profile for RPA-T191D (panel a ). These data show defined configurational states driven by the phosphocode. For all these experiments, representative data from a minimum of three independent experiments are shown.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Fluorescence, Mutagenesis, Mass Spectrometry, Structural Proteomics

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Structural model of the domains of RPA modeled using AlphaFold. The positions of phosphorylation (T191 and S384 in RPA70 and S23 and S29 in RPA32 are depicted. Red dotted boxes denote the positions probed here through phosphomimetic substitutions. b Circular dichroism (CD) spectra of RPA and the RPA phosphomimetic variants show no significant differences in the overall secondary structures. c ssDNA binding activity of RPA and RPA phosphomimetic variants were measured using fluorescence anisotropy and a fluorescein-labeled (dT) 35 oligonucleotide. The ssDNA binding of all three RPA proteins are stoichiometric due to the high-affinity ssDNA interactions of RPA. Stopped flow analysis of RPA-ssDNA interactions were performed by following the change in intrinsic Trp fluorescence changes as a function of increasing (dT) 35 concentrations. Data for ( d) . RPA 2D and e . RPA 3D are shown. Data were fit to single exponentials and ( f ) the k obs values plotted again ssDNA concentration yields similar k ON values for ssDNA binding for RPA proteins (RPA = 1.08 ± 0.2 x 10 8 M −1 s −1 , RPA 2D = 1.06 ± 0.2 x 10 8 M −1 s -1 , RPA 3D = 0.85 ± 0.1 x 10 8 M −1 s −1 ). g–j Mass photometry analysis of RPA or the RPA phosphomimetic variants on (dT) 50 ssDNA were performed at either 1:1 or 4:1 molar ratios of RPA:DNA. Predominantly single RPA-bound ssDNA complexes are observed at 1:1 ratios, and both 1:1 and 2:1 RPA-bound complexes are observed at higher ratios. However, for RPA 3D there is a higher fraction of the 2:1 complex. k Size exclusion chromatography analysis of RPA and the RPA phosphomimetics in the absence or presence of longer (dT) 97 ssDNA substrates shows similar profiles for RPA and RPA 2D interactions. However, RPA 3D forms complexes that are much larger, suggesting more RPA 3D molecules bound the ssDNA. In these experiments, a three-fold excess of RPA or RPA-phophomimetic variants were used to drive the assembly of multiple RPA molecules on the DNA. For all these experiments, representative data from a minimum of three independent experiments are shown. Error bars, where appropriate, are shown and represent St. Dev. values from n ≥ 3 independent experiments.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Phospho-proteomics, Circular Dichroism, Binding Assay, Activity Assay, Fluorescence, Labeling, Concentration Assay, Size-exclusion Chromatography

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Western blot analysis of in vitro kinase assay of recombinant RPA (150 nM or 250 nM) incubated with DNA-PK for 5 min. Blots probed with indicated antibodies. Data represent three to four independent experiments. b Quantitation of blots shown in ( a ). and phospho-S4/8 RPA32 levels were normalized to RPA70 control and expressed as fold over WT-RPA. * p = 0.0185 and ** p = 0.0036 (unpaired two-tailed t test). Error bars denote SEM of four independent experiments. c Western blot analysis of in vitro kinase assay of recombinant RPA (250 nM) incubated with DNA-PK for 5 min. d Quantitation of blots shown in c. and phospho-S4/8 RPA32 levels were normalized to RPA70 control and expressed as fold over WT-RPA. * p = 0.0337 and *** p = 0.0001 (unpaired two-tailed t test). Error bars denote SEM of three (T191D) or four independent experiments. e Western blot analysis of in vitro kinase assay of recombinant RPA (250 nM) incubated with DNA-PK for 5 min in the presence and absence of (dT) 97 ssDNA (50 nM). Blot is representative of three independent experiments. f Model depicts the positive feedback loop between RPA and the cell cycle-specific kinases that phosphorylate RPA. It remains unclear if the activity of kinases is regulated by RPA through a direct effect on their catalytic activity or through modulation of mediators such as WEE1. The OB-domains A, B, C, D, E are depicted in gray. The two protein interaction domains (F and wh) are colored purple and green. The disordered N-terminus of RPA32 is shown as a black line. g Model illustrates the release of the N-terminus of RPA32 (black line) along with the F and Wh domains upon cell cycle-specific priming phosphorylation at T191, S23, and S29 sites by CDK1 kinase. The phosphocode primes RPA32 for efficient downstream hyperphosphorylation by kinases such as DNA-PK in response to DNA damage. Thus, the structural re-organization induced by cell cycle-specific priming phosphorylation of both RPA70 and RPA32 works synergistically and is crucial for hyperphosphorylation of RPA32 in response to DNA damage. All molecular weight markers in the figure denoted in kDa.

Article Snippet: The following phospho-specific antibodies diluted in TBS-T with 1% milk were incubated overnight at 4 °C: pS384-RPA70 (custom antibody synthesized by Genscript described previously ), pS10-Histone H3 (Cell Signaling, 53348), pS4/S8-RPA32 (1:2000; Bethyl, A-300-245A), pS317-Chk1 (1:1000, Cell Signaling,), pS345-Chk1 (1:1000; Cell Signaling, 2341), S15-p53 (1:1000; Cell Signaling, 9284) or diluted with 5% milk: pY15-CDK1 (1:1000; Santa Cruz Biotechnology, sc136014) and pS33-RPA32 (1:1000; Bethyl, A300-246A).

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Incubation, Quantitation Assay, Control, Two Tailed Test, Activity Assay, Phospho-proteomics, Molecular Weight