WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress

Abstract

Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability.

Keywords: WRNIP1; genome instability; replication fork arrest; replication fork degradation.

Figure 1. Loss of WRNIP1 leads to nascent DNA strand degradation after HU‐induced replication stress

1. Western blot analysis showing the expression of the WRNIP1 protein in wild‐type cells (shWRNIP1^WT) and WRNIP1‐deficient (shWRNIP1) or mutant (shWRNIP1^T294A) cells. MRC5SV fibroblasts were used as a positive control. The membrane was probed with an anti‐FLAG or anti‐WRNIP1. GAPDH was used as a loading control. Below each lane of the blot the ratio of WRNIP1 protein to total protein, then normalized to MRC5SV, is reported.

2. Experimental scheme of dual labelling of DNA fibres in shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells. Cells were pulse‐labelled with CldU and then subjected to a pulse‐labelling with IdU.

3. Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks was measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t‐test).

4. Cells were treated as in (B). For each replication origin, the length of the right‐fork signal was measured and plotted against the length of the left‐fork signal. A schematic representation of symmetric and asymmetric forks is given. If the ratio between the left‐fork length and the right‐fork length deviated by more than 33% from 1 (that is, outside the violet dashed lines in the graphs), the fork was considered asymmetric. The percentage of asymmetric forks was calculated for all cell lines. N = number of forks counted for each cell line. R represents linear correlation coefficient.

5. Experimental scheme of dual labelling of DNA fibres in shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells. Cells were pulse‐labelled with CldU, treated with 4 mM HU and then subjected to a pulse‐labelling with IdU.

6. Graphs show the percentage of red (CldU) tracts (stalled forks) or red‐green (CldU‐IdU) contiguous tracts (restarting forks) in the cells. Means are shown, n = 3. Error bars represent standard error (*P < 0.05; **P < 0.01; Student's t‐test). Representative DNA fibre images are shown. Scale bar, 10 μm.

7. Experimental scheme of dual labelling of DNA fibres in shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells. Cells were sequentially pulse‐labelled with CldU and IdU as indicated, then treated or not with 4 mM HU.

8. Representative IdU tract length distributions in all cell lines under unperturbed conditions (top graph) or after HU treatment (bottom graph). Median tract lengths are given in parentheses. See also Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are shown. Scale bar, 10 μm.

Source data are available online for this figure.

Figure 2. Inhibition of MRE11 exonuclease activity prevents nascent DNA strand degradation after replication stress

1. Experimental scheme of dual labelling of DNA fibres in wild‐type cells (shWRNIP1^WT) or WRNIP1‐deficient cells (shWRNIP1). Cells were sequentially pulse‐labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 μM mirin.

2. Representative IdU tract length distributions in shWRNIP1^WT (top graph) or shWRNIP1 cells (bottom graph) after treatment. Median tract lengths are given in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are shown. Scale bar, 10 μm.

Figure 3. Analysis of parental ssDNA formation and RAD51 destabilization at stalled replication forks

1. Evaluation of ssDNA accumulation at parental‐strand by immunofluorescence analysis in wild‐type (shWRNIP1^WT) or WRNIP1‐deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity was chemically inhibited with 50 μM mirin, alone or in combination with HU‐induced replication stress. After treatment, cells were fixed and stained with an anti‐IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines and error bars represent the mean ± SE; n = 3 (ns, not significant; **P < 0.01; ****P < 0.0001; two‐tailed Student's t‐test). Representative images are shown. DNA was counterstained with DAPI (blue).

2. Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1^WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti‐WRNIP1, anti‐MRE11 and anti‐RAD51 antibodies. Lamin B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (input) in the cells was determined with the relevant antibodies. Lamin B1 was used as a loading control. In the graph, the fold increase with respect to the wild‐type untreated of the normalized ratio of the chromatin‐bound RAD51 (or MRE11)/total RAD51 (or MRE11) is reported for each cell line.

3. Analysis of DNA–protein interactions between ssDNA and endogenous RAD51 in shWRNIP1^WT and shWRNIP1 cells by in situ PLA assay. Experimental design used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti‐IdU antibody without denaturing the DNA to specifically detect parental‐strand ssDNA and subjected to PLA assay as described in the Materials and Methods section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51, respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows data presented as mean ± SE of the number of PLA spots per cell from three independent experiments (ns, not significant; **P < 0.01; two‐tailed Student's t‐test); n = 3.

4. Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU co‐immunoprecipitation (CldU‐IP). shWRNIP1^WT or shWRNIP1 cells were pulse‐labelled with CldU, then fixed or treated with HU. Cells were cross‐linked, and the nuclear extracts were isolated (input) and subjected to CldU‐IP using an anti‐CldU antibody (CldU‐IP). The membranes were probed with the anti‐WRNIP1 or anti‐RAD51 antibodies. After stripping, the membranes were probed with an anti‐MRE11 antibody. Lamin B1 and GAPDH were used as loading controls (input). Ponceau S was used as a loading control of CldU‐IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitated DNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti‐CldU antibody. The graph shows the normalized ratio of the proteins co‐immunoprecipitated with CldU (CldU Co‐IP proteins)/the total of labelled DNA immunoprecipitated with CldU (CldU‐IP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.

Source data are available online for this figure.

Figure 4. RAD51 protects nascent DNA strand from degradation after fork stalling in the absence of WRNIP1

1. Experimental scheme of pulse‐labelling of DNA fibres in wild‐type cells (shWRNIP1^WT) or WRNIP1‐deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.

2. Representative IdU tract length distributions in shWRNIP1^WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are reported. Scale bar, 10 μm.

3. Scheme of DNA fibre tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild‐type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.

4. Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild‐type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are given. Scale bar, 10 μm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti‐RAD51. Lamin B1 was used as a loading control.

Source data are available online for this figure.

Figure 5. WRNIP1 stabilizes RAD51 on stalled forks

1. Co‐immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG‐WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG IP) using anti‐FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti‐FLAG, anti‐RAD51 and anti‐BRCA2 antibodies, respectively. Whole‐cell extracts were analysed (input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control.

2. Analysis of protein–protein interactions between WRNIP1 and endogenous RAD51 in wild‐type (shWRNIP1^WT) or WRNIP1‐mutant (shWRNIP1^T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG‐Tag and RAD51 were used to reveal FLAG‐WRNIP1 or endogenous RAD51, respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell ± SE. Error bars represent standard error (ns, not significant; two‐tailed Student's t‐test); n = 3.

3. Experimental scheme of pulse‐labelling of DNA fibres in wild‐type cells (shWRNIP1^WT) or WRNIP1‐deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.

4. Representative IdU tract length distributions in shWRNIP1^WT/siBRCA2 or shWRNIP1^siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are reported. Scale bar, 10 μm. Western blot shows BRCA2 depletion in shWRNIP1^WT and shWRNIP1 cells. The membrane was probed with an anti‐BRCA2 or anti‐WRNIP1. GAPDH was used as a loading control.

5. Experimental scheme of pulse‐labelling of DNA fibres in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1^siCtrl) or FBH1 siRNA (shWRNIP1^siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.

6. Representative IdU tract length distributions in shWRNIP1^siCtrl or shWRNIP1^siFBH1 cells with or without HU treatment. Representative DNA fibre images are reported. Scale bar, 10 μm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti‐FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test.

7. Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1^siCtrl) or FBH1 siRNA (shWRNIP1^siFBH1), and 48 h treated or not with HU for 4 h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti‐FBH1 and anti‐RAD51 antibodies. Lamin B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/Lamin B1 signal (chromatin) is reported below each lane.

Source data are available online for this figure.

Figure 6. Loss of WRNIP1 or its ATPase activity results in DNA damage accumulation and enhanced chromosomal instability in response to fork stalling

1. Analysis of DNA damage accumulation. Wild‐type (shWRNIP1^WT), WRNIP1‐deficient (shWRNIP1) or mutant (shWRNIP1^T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to γ‐H2AX immunofluorescence. Graph shows data presented as mean of γ‐H2AX‐positive cells ± SE from three independent experiments; n = 3 (*P < 0.1; **P < 0.01; two‐tailed Student's t‐test). Representative images of nuclei showing the different number of foci per nucleus are reported.

2. Analysis of DNA breakage accumulation. shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells were treated as in (A), then subjected to alkaline comet assay. Graph shows data presented as mean tail moment ± SE from three independent experiments; n = 3 (*P < 0.1; **P < 0.01; two‐tailed Student's t‐test). Representative images are shown.

3. Evaluation of cell death. shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells ± SE from three independent experiments; n = 3 (*P < 0.1; **P < 0.01; two‐tailed Student's t‐test). Representative images of double‐staining of viable (green) and dead (red) cells are shown.

4. Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1^WT, shWRNIP1 and shWRNIP1^T294A cells were treated or not with 4 mM HU, then left to recover for 16 h in drug‐free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in Appendix Supplementary Materials and Methods. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines and error bars represent the mean ± SE (ns, not significant; **P < 0.01; two‐tailed Student's t‐test). Representative Giemsa‐stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.

5. Experimental scheme of the chromosomal aberration analysis is given. The experiment was carried out as in (D) but cells were pre‐treated or not with 50 μM mirin. Dot plot shows the effect of mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines and error bars represent the mean ± SE (ns, not significant; **P < 0.01; two‐tailed Student's t‐test). Representative Giemsa‐stained metaphases of shWRNIP1 cells treated with mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.

6. Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Forty‐eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in Appendix Supplementary Materials and Methods. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti‐FBH1. GAPDH was used as a loading control. Horizontal black lines and error bars represent the mean ± SE (**P < 0.01; two‐tailed Student's t‐test).

Source data are available online for this figure.

Figure 7. Schematic model for the role of WRNIP1 at stalled forks

WRNIP1 interacts with the BRCA2/RAD51 complex and stabilizes RAD51 on ssDNA at stalled forks, counteracting the dissolution of the RAD51 filament by FBH1. After stalled fork stabilization, the ATPase activity of WRNIP1, in collaboration with other proteins, could be required for stimulating the restart of DNA synthesis, which ensures genome stability. Loss of WRNIP1 or its catalytic activity leads to DNA damage accumulation and enhanced chromosomal instability. See the text for more details.