Journal: bioRxiv

Article Title: Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures

doi: 10.1101/2025.02.21.639251

Figure Lengend Snippet: A. Representative 2D maximum projections of 3D-SIM images of an early and mid S-phase nucleus after labelling with the standard pulsing scheme and post-fixation PCNA immunofluorescence staining, scale bar = 5µm. B. Bar plot showing the mean correlation coefficient (Pearson’s R value with no threshold) between indicated image channels, as determined by FIJI plugin Coloc 2, n = 4. Error bars represent SD. C. Examples of nuclear areas containing individual replication events, with arrows indicating inferred directionality, scale bar = 500nm. For each area, an EdU only, Cy3-xx-dUTP only, PCNA only and merged channel image are shown below. D. Bar plot showing the mean nuclear integrated intensities (a.u.) of pH2AX and pRPA for cells treated as described in 1E and 1F, respectively. Three replicates per condition, error bars represent SD. E. Representative 2D maximum projections of 3D-SIM images of cells after three different treatments: EdU for 30-minutes only, EdU for 30-minutes and Cy3-xx-dUTP for 30 (4 + 26)-minutes or 2-hours HU treatment prior to labelling with EdU for 30-minutes followed by Cy3-xx-dUTP for 30 (4 + 26)-minutes. All conditions underwent post-fixation immunofluorescence staining of pH2AX, scale bars: 10µm. The nuclear perimeter is annotated with a white dashed line and a selected area (white square) is shown in the merged image, scale bars: 2µm. F. Representative 2D maximum projections of 3D-SIM images of cells after three different treatments: EdU for 30-minutes only, EdU for 30-minutes and Cy3-xx-dUTP for 30 (4 + 26)-minutes or 2-hours HU treatment prior to labelling with EdU for 30-minutes followed by Cy3-xx-dUTP for 30 (4 + 26)-minutes. All conditions underwent post-fixation immunofluorescence staining of pRPA, scale bars: 10µm. The nuclear perimeter is annotated with a white dashed line and a selected area (white square) is shown in the merged image, scale bars: 2µm.

Article Snippet: If immunostaining applied, fixed samples were incubated overnight at 4°C with a primary antibody: phospho-histone H2A.X [Ser139] (1:800, Cell Signalling Technology, #2577); phospho-RPA32 [S33] (1:2000, Bethyl Laboratories, #A300-246A); or PCNA (PC10) (1:400, Santa Cruz, sc-56) in blocking media.

Techniques: Immunofluorescence, Staining

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 1. Nuclear distribution of PCNA and RPA1 during hydroxyurea-induced replication stress. (A) Comparison of the expression level of BAC-tagged PCNA-mCherry and RPA1-EGFP versus their endogenous counterparts via Western blotting. (B) Representative timelapse airyscan images of RPA1-EGFP and PCNA-mCherry before and during HU-induced replication stress. Scale bar = 5 µm. (C) Same as (B), but in the presence of ATR inhibitor AZD6738 before and during HU treatment. Scale bar = 5 µm. Abbreviations: HU: hydroxyurea; AZD: AZD6738.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Comparison, Expressing, Western Blot

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 2. Dynamics of RPA1 and PCNA during HU-induced replication fork stalling and restart. (A) Representative time-lapse images of RPA1 and PCNA before, during, and after HU treatment. Arrows indicate timepoints of HU addition and washout. Scale bar = 5 µm. (B) Same as (A), but with inhibition of ATR (3 µM AZD6738) throughout the experimental period. (C) Normalized intensity of PCNA and RPA1 at replication foci during HU-induced replication fork stalling and restart, with or without ATR inhibition. The maximum intensity of PCNA/RPA at replication foci is normalized to 1. (D) Fraction of PCNA and RPA1 bound at replication foci during HU-induced replication fork stalling and restart, with or without ATR inhibition, relative to the total nuclear intensity of PCNA/RPA1, which is normalized to 1. (E) Estimated number of PCNA homotrimer and RPA heterotrimer complexes engaged at replication foci with or without ATR inhibition. (F) Estimated number of nucleotides covered by RPA heterotrimers with or without ATR inhibition. For HU only: n = 17 cells; for HU + AZD: n = 10 cells. Abbreviations: HU: hydroxyurea; AZD: AZD6738.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Inhibition

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 3. Detailed overview for the measurement and quantification of PCNA and RPA1 engaged at replication foci during fork stalling and restart. (A) Regions of interest applied for analysis. ‘A’ represents the cell nucleus (white lining), ‘B’ represents a region within the nucleus without replication foci (blue ellipse), and ‘C’ represents noise outside of cells (orange square). (B) Constants and formulas for the calculation of variables used for quantifying RPA1 and PCNA kinetics. (C) Mean intensity of diffusing RPA1-EGFP/mPCNA-mCherry within region ‘B’, calculated as shown in the formula for Dt.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques:

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 4. Exchange of PCNA and RPA1 at replication foci during unperturbed and stalled replication. (A) Representative timelapse images depicting simultaneous FRAP of RPA1 and residual PCNA co-localized at stalled replication forks under nucleotide depletion. (B) Same as (A), but under conditions of ATR inhibition. (C) Replication timelapse images of PCNA FRAP at replication foci during unperturbed replication (upper panel) or in the presence of ATR inhibitor AZD6738. (D) FRAP curves of PCNA under the following conditions: untreated, HU alone, AZD alone, HU + AZD. The mean intensity is normalized as described in Figure S5. (E) Contribution of distinct PCNA fractions (freely diffusing [F1] and replisome-bound [F2]) to the FRAP curve under nucleotide depletion, as determined via fitting of two single exponential curves. (F) Same as (E), but in the presence of both AZD6738 and HU. (G) Recovery of the replisome-bound fraction of PCNA under HU treatment, as derived based on (E). (H) Recovery of the replisome-bound fraction of PCNA under HU treatment, as derived based on (F). (I) Comparison of PCNA recovery under the following conditions: untreated, AZD alone, HU alone, HU + AZD. The contribution of freely diffusing PCNA has been subtracted from the HU and HU + AZD curves, as per (G,H). (J) Number of PCNA complexes at a single replication fork, recovered after photobleaching. (K) Enlarged view of HU and HU + AZD curves from (J). (L) FRAP curves of RPA1 at replication foci under HU alone and HU + AZD. (M) Number of RPA complexes at a single replication fork recovered after photobleaching under HU alone and HU + AZD. For HU: n = 11 cells; for HU + AZD: n = 16 cells; for AZD (PCNA only): n = 13 cells; untreated (PCNA only): n = 15 cells. Abbreviations: HU: hydroxyurea; AZD: AZD6738.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Inhibition, Derivative Assay, Comparison

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 5. Influence of ATM activity on RPA1 and PCNA dynamics during HU-induced replication fork stalling and restart under conditions of ATR inhibition. (A) Representative time-lapse images of RPA1 and PCNA before, during, and after 10 mM HU treatment under combined ATM (10 µM Ku55933) and ATR (3 µM AZD6738) inhibition. Arrows indicate timepoints of HU addition and washout. Scale bar = 5 µm. (B) Normalized intensity of PCNA and RPA1 at replication foci during HU-induced replication fork stalling and restart under ATM inhibition with or without ATR co- inhibition. The maximum intensity of PCNA/RPA1 engaged at replication foci is normalized to 1. (C) Fraction of PCNA and RPA1 bound at replication foci during HU-induced replication fork stalling and restart under ATM inhibition with or without ATR inhibition, relative to the total nuclear intensity of PCNA/RPA1, which is normalized to 1. (D) Normalized intensity of PCNA and RPA1 at replication foci during HU-induced replication fork stalling and restart under ATR inhibition with or without ATM co-inhibition. The maximum intensity of PCNA/RPA1 engaged at replication foci is normalized to 1. (E) Fraction of PCNA and RPA1 bound at replication factories during HU-induced replication fork stalling and restart under ATR inhibition with or without ATM inhibition, relative to the total nuclear intensity of PCNA/RPA1, which is normalized to 1. (F) Same as (B,D), but with or without combined ATR + ATM inhibition. (G) Same as (C,E), but with or without combined ATR + ATM inhibition. (H) Estimated number of PCNA homotrimer and RPA heterotrimer complexes engaged at replication foci during HU-induced fork stalling and subsequent restart. (I) Estimated number of nucleotides covered by RPA heterotrimers under combined ATM and ATR inhibition. Dashed orange lines indicate the timepoints of HU addition and wash-out. For HU + AZD + KU, n = 10 cells; for HU + KU: n = 19 cells. Abbreviations: HU: hydroxyurea; AZD: AZD6738; KU: Ku55933.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Activity Assay, Inhibition

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 6. Dynamics of PCNA and RPA1 during hydroxyurea-induced replication stress in HeLa, DU145, and PC3 cell lines. (A) Normalized intensity of PCNA and RPA1 during, before, and after HU treatment. (B) Same as (A), but under conditions of ATM inhibition (10 µM Ku55933). (C) Same as (A), but under conditions of ATR inhibition (3 µM AZD6738). (D) Same as (A), but under combined ATR and ATM inhibition. Dashed green lines indicate the timepoints of HU addition and washout. Data are presented as the mean ± SD. For HeLa: n = 11 cells (HU), n = 16 cells (HU + AZD), n = 19 cells (HU + KU), n = 10 cells (HU + AZD + KU); for DU145: n = 11 cells (HU), n = 10 cells (HU + AZD), n = 10 cells (HU + KU), n = 18 cells (HU + AZD + KU); for PC3: n = 18 cells (HU), n = 10 cells (HU + AZD), n = 13 cells (HU + KU), n = 15 cells (HU + AZD + KU). Abbreviations: HU: hydroxyurea; AZD: AZD6738; KU: Ku55933.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Inhibition

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 7. Dynamics of RAD18 during hydroxyurea-induced replication fork stalling and restart. (A) Representative time-lapse images of RAD18 and PCNA before, during, and after HU treatment. Arrows indicate timepoints of HU addition and washout. Scale bar = 5 µm. (B) Normalized intensity of RAD18 and PCNA during HU-induced replication fork stalling and restart, with or without ATR inhibition (3 µM AZD6738). The maximum intensity of PCNA/RAD18 foci is normalized to 1. For HU: n = 17 cells; for HU + AZD: n = 12 cells. (C) Normalized intensity of single RAD18 and PCNA foci before and after HU addition (max intensity = 1). Single foci were tracked using SPARTACUSS, and a representative kymogram is shown. This panel presents a case where RAD18 accumulates at a replication focus, while PCNA dissociates after HU treatment. (D) Same as (C), but under conditions of ATR inhibition. (E) Same as (C), but this panel presents a case where RAD18 is already present at the replication focus and dissociates from the replication focus in parallel to PCNA upon HU addition. (F) Same scenario as shown in (E), but under conditions of ATR inhibition. Dashed green lines indicate timepoints of HU addition and removal. Abbreviations: HU: hydroxyurea; AZD: AZD6738.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: Inhibition

Journal: International journal of molecular sciences

Article Title: In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell.

doi: 10.3390/ijms26020667

Figure Lengend Snippet: Figure 8. Visual summary of PCNA and RPA dynamics during replication fork stalling and restart.

Article Snippet: After blocking, membranes were incubated with mouse primary antibodies against PCNA (PC10) (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and RPA70/RPA1 (MBS600132, MyBioSource, San Diego, CA, USA) overnight at 4 ◦C, followed by incubation with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio, Lincoln, NE, USA) at room temperature for 1 h. Bands were visualized using an Odyssey Infrared Imaging System (LICORbio) and saved as TIF images.

Techniques: