Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

Figure Lengend Snippet: a. A 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 T191 residue. MS analysis was performed using endogenous RPA70 immunoprecipitated from HCT116 cells arrested in mitosis using 50 ng/mL nocodazole for 18 hrs. Sequence alignment of RPA70 (RFA1) protein reveals a conserved SP/TP motif. (*) indicate positions which have a single, fully conserved residue, (:) indicates conservation between groups of strongly similar properties, and (.) indicates conservation between groups of weakly similar properties. d. Mass spectrum of the RPA70 tryptic peptide showing phosphorylation at the T191 residue. Reactions were extracted from in vitro kinase assay of recombinant human RPA incubated with Cdk1/Cyclin B complex followed by MS-MS analysis.

Article Snippet: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

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

Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

Figure Lengend Snippet: a. Cartoon depicts hyperphosphorylation of the N- terminus of RPA32 in response to DNA damage. It is unclear as to how disruption of T191-RPA70 phosphorylation affects 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 21hrs was determined by the Caspase3/7 glo assay. Bar graph depicts data corrected for background absorbance and plotted as fold change over vehicle (veh) control (0.1% DMSO). Error = SEM. Mean of three independent experiments are plotted. 3 to 4 wells were assayed per experiment. Statistical significance was determined using an unpaired two-tailed t -test: *** p = 0.0042. c. Western blot analysis of cells treated briefly with 20 ng/mL SN-38 for 90 min or vehicle (0.1% DMSO). Blots were probed with the indicated antibodies with Tubulin and Vinculin as loading controls. Blots are representative of three independent experiments. d. Cells synchronized with double thymidine block were released and at 7 hrs post-release, cells in G2 were treated with 100 μM etoposide for 45 min. Blots were probed with the indicated antibodies with Tubulin as loading control. Blots are representative of three independent experiments.

Article Snippet: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

Techniques: Disruption, Activity Assay, Glo Assay, Control, Two Tailed Test, Western Blot, Blocking Assay

Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

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. b. Circular dichroism (CD) spectra of RPA, RPA-T191A, and T191D does not show significant differences in the overall secondary structures. 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. 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±0.2×10 8 M -1 s -1 , RPA- T191A = 0.86±0.3×10 8 M -1 s -1 , RPA-T191D = 1.06±0.2×10 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 show 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: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

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

Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

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 F-E OB domains and the wh domain are denoted. The disordered N-terminus of RPA32 and two protein interaction domains (F and wh) are marked by the dashed squares. 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. c. XL-MS analysis of the RPA-T191D,S23D,S29D triple phosphomimetic mutant shows pattern 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-T191D,S23D,S29D show differences that are marginally higher than RPA. This observation is different that 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: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

Techniques: Fluorescence, Mutagenesis, Mass Spectrometry

Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

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 position probed here through phosphomimetic substitutions. b. Circular dichroism (CD) spectra of RPA and the RPA phosphomimetic variants shows 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. 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-S23D,S29D and e. RPA- T191D,S23D,S29D 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±0.2×10 8 M -1 s -1 , RPA-S23D,S29D = 1.06±0.2×10 8 M -1 s -1 , RPA-T191D,S23D,S29D = 0.85±0.1×10 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-T191D,S23D,S29D 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 show similar profiles for RPA and RPA-S23D,S29D interactions. However, RPA-T191D,S23D,S29D for complexes that are much larger suggesting more RPA molecules bound the ssDNA for this RPA phosphomimetic variant. In these experiments, a three-fold excess of RPA or RPA- phophomimetics 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: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

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

Journal: bioRxiv

Article Title: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

doi: 10.1101/2025.01.16.633180

Figure Lengend Snippet: Western blot analysis of in vitro kinase assay of recombinant RPA (150nM or 250nM) incubated with 80 units of DNA-PK for 5 min. Blots were probed with the indicated antibodies. Data represent three independent experiments. b. Model depicts the positive feedback loop between RPA and the cell cycle-specific kinases that phosphorylate RPA. Domains A, B, C, D, E are depicted. 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 protein interaction domains (F and wh) are also shown. The disordered N-terminus of RPA32 is shown as a black line. c. 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 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 work synergistically and is crucial for hyperphosphorylation of RPA32 in response to DNA damage.

Article Snippet: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4°C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), Cdk1 (1:1000; Santa Cruz Biotechnology, sc-54), Wee1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-24).

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Incubation, Activity Assay

Journal: bioRxiv

Article Title: Genomic and transcriptomic profiling of high-risk bladder cancer reveals diverse molecular and microenvironment ecosystems

doi: 10.1101/2024.12.21.629010

Figure Lengend Snippet: A. FGFR3 mRNA levels across DepMap cell lines classified as having high or low expression of Subtype 4 gene program markers. (p-value was calculated from a two-sided Kruskal-Wallis test) B. Volcano plot showing differences in IC50 between cell-lines with high vs low Subtype 4 gene program score on the x-axis and -Log10 adjusted p-value for each comparison on the y-axis. Each point represents a unique chemical perturbation (Left). Drugs with increased sensitivity in cell-lines that have high expression of Subtype 4 gene program (Right). C. MYC and FGFR3 protein levels in UMUC3 or UMUC9 cell-lines overexpressing GFP controls or FGFR3 S249C mutations. (Vinculin was used as loading control for the cytoplasmic fraction, RPA32 was used as loading control for the nuclear fraction) (n=2) D. Survival curves for UMUC3 and UMUC9 cell-lines overexpressing GFP controls or FGFR3 S249C mutations treated with MEK inhibitor Trametinib or MYC inhibitor for 7 days. (n=3).

Article Snippet: Protein expression was verified using western blotting with these antibodies: anti-ERBB2 (Cell Signaling Technologies, 2165), anti-FGFR3 (Cell Signaling Technologies, 4574), anti-RPA32 (Cell Signaling Technologies, 35869), anti-Tubulin (Sigma, T5168), anti-Vinculin (Invitrogen, MA5-11690), anti- MYC (Invitrogen, 13-2500).

Techniques: Expressing, Comparison, Control

Journal: Cell Death Discovery

Article Title: Amodiaquine ameliorates stress-induced premature cellular senescence via promoting SIRT1-mediated HR repair

doi: 10.1038/s41420-024-02201-1

Figure Lengend Snippet: A , B HFF1 cells were treated with 0, 1, 5, and 10 μM AQ for 48 h. The expression levels of SIRT1, SIRT2, SIRT3, and SIRT6 were determined by Western blotting ( A ); and SIRT1 mRNA were measured by qRT-PCR ( B ). C HFF1 cells were treated with 5 μg/ml CHD alone or in combination with AQ for 0, 6, 12, 24 and 48 h. Subsequently, the relative mRNA of SIRT1 was evaluated by qRT-PCR. D HFF1 cells were pretreated with AQ 24 h prior to transfection with the luciferase reporter plasmids. The relative luciferase activity normalized to Renilla luciferase activity is presented. E Cell lysates were immunoprecipitated with anti-SIRT1 antibody after treatment with AQ for 48 h, followed by immunoblotting with anti-NBS1 and anti-Rad51 antibodies. F HFF1 cells were irradiated with 4 Gy of X-ray after a 24-h period of AQ treatment. Cells were immunostained for Rad51 foci (green) at 4 h post irradiation. G The number of Rad51 foci per cell. Significance markers: * p < 0.05 compared to control; n = 3.

Article Snippet: After blocking with 5% BSA, membranes were incubated with the following primary antibodies, all diluted to 1:1000: p21 WAF1 (#37543), p16 ink4a (#18769), pDNA-PKcs (#68716), DNA-PKcs (#4602), Ligase IV (#14649), Ku80 (#2180), Ku70 (#4588), BRCA2 (#10741), BRCA1 (#14823), CtIP (#9201), Mre11 (#4895), Rad50 (#3427), pNBS1 (#3001), NBS1 (#14956), Rad54 (#15016), Rad51 (#8875), RPA2 (#35869), SIRT1 (#9475), SIRT2 (#12650), SIRT3 (#5490), SIRT6 (#12486), Histone Deacetylase (HDAC) Antibody Sampler Kit (#9928) and β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Immunoprecipitation, Irradiation, Control

Journal: Cell Death Discovery

Article Title: Amodiaquine ameliorates stress-induced premature cellular senescence via promoting SIRT1-mediated HR repair

doi: 10.1038/s41420-024-02201-1

Figure Lengend Snippet: A Western blotting of the SIRT1 expression in HFF1 cells transfected with control or Rad51 siRNA. B , C The SIRT1-depleted cells were pretreated with AQ for 24 h prior to 8 Gy of X-ray and further incubated for 8 h, then the cells were collected for neutral comet assay. The comet assay images ( B ) and quantification of tail moment ( C ) were shown. D , E The SIRT1-depleted cells were pretreated with AQ for 24 h prior to 4 Gy of X-ray. Immunofluorescent staining for γH2AX foci (red) were performed 8 h post irradiation. The representative images ( D ) and the number of γH2AX foci per cell ( E ) were presented. The SIRT1-depleted cells were treated with indicated conditions and subjected to HR efficiency evaluation by using our detecting system ( F ) and DR-GFP reporter assay ( G ). Significance markers: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to control; n = 3.

Article Snippet: After blocking with 5% BSA, membranes were incubated with the following primary antibodies, all diluted to 1:1000: p21 WAF1 (#37543), p16 ink4a (#18769), pDNA-PKcs (#68716), DNA-PKcs (#4602), Ligase IV (#14649), Ku80 (#2180), Ku70 (#4588), BRCA2 (#10741), BRCA1 (#14823), CtIP (#9201), Mre11 (#4895), Rad50 (#3427), pNBS1 (#3001), NBS1 (#14956), Rad54 (#15016), Rad51 (#8875), RPA2 (#35869), SIRT1 (#9475), SIRT2 (#12650), SIRT3 (#5490), SIRT6 (#12486), Histone Deacetylase (HDAC) Antibody Sampler Kit (#9928) and β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Control, Incubation, Neutral Comet Assay, Single Cell Gel Electrophoresis, Staining, Irradiation, Reporter Assay