MRNIP limits ssDNA gaps during replication stress

Abstract

Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.

Graphical Abstract

Figure 1.

Impaired fork reversal drives MRE11-dependent post-replicative ssDNA gaps in MRNIP KO cells. (A) Top: DNA fibre assay schematic. WT and MRNIP KO HeLa cells were labelled with CldU for 20 min, then labelled with IdU for 60 min in the presence of PBS or 150 μM cisplatin. Cells were then treated −/+ S1 nuclease and DNA fibre assays were performed. IdU tract length was measured using ImageJ. (B) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with 1 μM Olaparib, then nascent DNA was labelled with CldU and IdU −/+S1 nuclease. (C) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with DMSO, 1 μM Olaparib, 150 μM cisplatin, or 1 μM Olaparib + 150 μM cisplatin, then nascent DNA was labelled with CldU and IdU −/+S1 nuclease. (D) Representative examples of DNA fibres from Olaparib + cisplatin-treated WT and MRNIP KO cells. (E, F) WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or validated siRNAs targeting either SMARCAL1 or ZRANB3. After 48 h, cells were lysed and SMARCAL1 depletion was confirmed by Western blotting (E). RNA was isolated and ZRANB3 mRNA levels were quantified by qRT-PCR and normalized to GAPDH (F). (G) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or siRNAs targeting SMARCAL1 or ZRANB3. After 48 h, nascent DNA was labelled with CldU, then IdU and cisplatin −/+ S1 nuclease. (H) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with 1 μM Olaparib and either DMSO or 25 μM PFM39, then nascent DNA was labelled with CldU, followed by IdU labelling in the presence of 100 μM cisplatin −/+ S1 nuclease. Experiments were performed three times independently and were confirmed by at least two members of the laboratory. Results are displayed as dot plots with indicated median values and analysed via one-way ANOVA. **P< 0.01, ****P< 0.0001

Figure 2.

ssDNA gaps in MRNIP KO cells are driven by replication repriming. (A) WT and MRNIP KO HeLa cells were transfected with either a non-targeting control siRNA, or a validated siRNA targeting PRIMPOL. After 48 hrs, whole cell extracts were generated and resolved by Western blotting with the indicated antibodies. (B) Top: DNA fibre assay schematic. WT and MRNIP KO HeLa cells were transfected as in (A), then treated with Olaparib and cisplatin as in the indicated schematic, followed by S1 nuclease treatment. Results are displayed as dot plots with indicated median values. (C) WT and MRNIP KO HeLa cells were transfected with control siRNA or an siRNA targeting PRIMPOL. After 24 h, cells were incubated for 16 h with 25 μM IdU, then treated with 100 μM cisplatin or 1 μM Olaparib alone or in combination for 4 h. Cells were fixed and stained with a BrdU antibody that cross-reacts with IdU. IdU-positive cells were counted, and results displayed as a proportion of the total number of cells. (D) Representative images of native IdU staining in Olaparib and cisplatin-treated MRNIP KO cells, following transfection with control siRNA or PRIMPOL siRNA. (E) WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or an siRNA targeting PRIMPOL. Forty-eight hours later, cells were treated with either DMSO, 100 μM cisplatin, 1 μM Olaparib, or both drugs for 16 h, then were fixed and stained with a γH2AX antibody, and counterstained with DAPI. Cells with more than 5 foci were counted and results plotted as a percentage of the total number of cells. (F) WT and MRNIP KO cells were transfected as in (A). After 48 h, cells were replated and treated with the indicated concentrations of Olaparib. After 12 days, surviving colonies were counted and results normalised to untreated controls. All experiments were performed three times independently and analysed using one-way ANOVA. *P< 0.05, ***P< 0.001, ****P< 0.0001

Figure 3.

Fork reversal and REV1 limit ssDNA gaps and DNA damage markers in MRNIP KO cells. (A) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or siRNAs targeting either SMARCAL1 or ZRANB3. After 48 h, cells were treated with 1 μM JH-RE-06 (REV1 inhibitor), then nascent DNA was labelled with CldU for 20 min, followed by IdU for 60 min. ssDNA was then digested with S1 nuclease. IdU tract length was measured using ImageJ. IdU tract length was measured using ImageJ. (B–D) WT and MRNIP KO cells were transfected with a control siRNA, or an siRNA targeting SMARCAL1. Forty-eight hours later, cells were treated with either DMSO or 1 μM JH-RE-06 for 16 hrs, then fixed and stained with antibodies against γH2AX (B) and 53BP1 (C), and counterstained with DAPI. Cells with more than 5 foci were counted and results plotted as a percentage of the total number of cells. Representative images are displayed in (D). All experiments were performed three times independently and analysed via one-way ANOVA. *P< 0.05, ****P< 0.0001.

Figure 4.

Post-replicative gap filling in MRNIP KO cells is mediated by UBC13 and REV1/Pol-ζ. (A) Parental U2OS cells, or Super RPA U2OS cells were lysed, and extracts were resolved by SDS-PAGE and probed via Western blotting with the indicated antibodies. (B) Top: Schematic of DNA fibre assay. Parental and Super RPA U2OS were treated with 1 μM Olaparib, then nascent DNA was labelled with CldU for 20 min, followed by labelling with IdU in the presence of 150 μM cisplatin for 60 min. ssDNA was then digested with S1 nuclease. IdU tract length was measured using ImageJ. (C) WT and MRNIP KO HeLa cells were treated with 1 μM Olaparib, then nascent DNA was labelled with CldU, followed by IdU labelling in the presence of 150 μM cisplatin to allow post-replicative gaps to form. Cells were then immediately lysed and DNA fibres spread (Control), or cells were treated with a combination of 4 mM HU and 25 μM PFM39 for 4 h, then mock-treated or S1 nuclease-treated prior to spreading. DNA fibre spreading and analysis was performed and the length of IdU tracts was measured using ImageJ. (D, E) MRNIP KO cells were transfected with the indicated siRNAs, and knockdowns were confirmed by Western blotting (D) or qRT-PCR (E). (F) MRNIP KO cells were transfected with the indicated siRNAs and then labelled and treated with PFM39 and HU as in (C) prior to S1 nuclease treatment and DNA fibre spreading and analysis. All experiments were performed three times independently. Data in (F) was confirmed independently by two laboratory members. Results are displayed as dot plots with indicated median values and analysed using one-way ANOVA. ****P< 0.0001.