flag-brca1 plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: RIF1-L phospho-SPKF peptide binds to tandem BRCT domain of BRCA1 in vitro (A) (B) Fluorescence polarisation analysis of binding affinities between BRCA1-BRCT domain and RIF1-L phospho-peptide with indicated treatment or mutations. (C) Crystal structure of RIF1-L phospho-peptide (stick presentation) in complex with BRCA1-BRCT domain (space-filling presentation), solved by X-ray diffraction. The phosphorus atom is represented in orange. Residues on RIF1-L phospho-peptide are labelled with pink text (S2265,K2267,F2268,K2269). Residue on BRCA-BRCT domain is labelled with green text (E1698). (D) Model of RIF1-L interaction with BRCA1. In RIF1-L protein presentation, purple bards indicate PP1-interacting motifs; pink box indicates Exon 31; red bar indicates the ‘S 2265 PKF’ motif. RIF1-L phosphoS 2265 PKF motif binds to the C-terminal tandem BRCT domains of BRCA1.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: In Vitro, Fluorescence, Binding Assay, Residue

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: RIF1-L isoform exhibits proximity with BRCA1 in vivo under replication stress (A) Representative images of RIF1-BRCA1 PLA foci in RPE-1 cells with indicated treatments. Scalebar: 10µm. (B) Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as (A). In this and all following Tukey box-and-whisker plots, box represents 1st to 3rd quartile, horizontal line inside the box represents median. p values are calculated by Mann-Whitney test for non-parametric distributions. n.s. p >0.05; * p ≤0.05; ** p ≤0.01; *** p ≤0.001; **** p ≤0.0001. (C) RIF1-BRCA1 PLA analysis in RPE-1 cells with indicated treatments. HU: 4mM 24hr; APH: 4µM 24hr; CPT: 100nM 24hr. (D) Schematic representation of RIF1-L Morpholinos (bright blue lines) targeting the splicing signals for RIF1-L Exon31 inclusion. The RIF1-L morpholinos were designed to inhibit spliceosome recruitment leading to the skipping of Exon 31 during pre-mRNA splicing. Treatment with RIF1-L Morpholinos is expected to prevent the generation of RIF1-L mRNA. (E) Gel analysis of RT-PCR-based analysis to distinguish RIF1-L and RIF1-S transcripts in Control and RIF1-L Morpholino-treated RPE-1 cells. The upper band corresponds to RIF1-L mRNA and the lower band corresponds to RIF1-S mRNA. Experimental procedure described in Fig.S3A. (F) Western blot analysis of total RIF1 protein expression in Control and RIF1-L Morpholino-treated RPE-1 cells. Mo: abbreviation for Morpholino. (G) RIF1-BRCA1 PLA analysis in Control and RIF1-L Morpholino-treated RPE-1 cells with indicated HU treatments. Experiment was repeated twice (see also Fig.S3C), and one representative result is shown.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: In Vivo, Whisker Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A Western blot analysis of 53BP1 depletion in RPE-1 cells. (B) RIF1-BRCA1 PLA analysis in Control or 53BP1-depleted RPE-1 cells with indicated treatments. HU: 4mM 24hr. Experiment was repeated twice (see also Fig.S4A), and one representative result is shown. (C) Experiment procedure to label S phase cells with EdU, followed by RIF1-BRCA1 PLA analysis. An asynchronous RPE-1 cell culture was pulse labelled with EdU for 15 min, and then treated with no or 4mM HU for 4hr. Cells were fixed at the end of HU treatment and subjected to RIF1-BRCA1 PLA procedures. (D) Left: representative images acquired from the experiment described in (C). unt: not treated with HU; HU: 4mM 4hr. Scalebar: 10µm. Right: quantification of RIF1-BRCA1 PLA foci per nucleus. Experiment was repeated twice (see also Fig.S4D), and one representative result is shown. (E) RIF1-BRCA1 PLA analysis in RPE-1 cells collected at indicated timepoints after removal of HU. Experiment was repeated twice (see also Fig.S4F), and one representative result is shown. (F) RIF1-BRCA1 PLA analysis in RPE-1 cells following indicated kinase inhibitor and HU treatments. HU: 4mM 24hr; ATRi (VE-821): 1µM 24hr; Chk1i (PF-477736): 1µM 24hr. Experiment was repeated twice (see also Fig.S4G), and one representative result is shown.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Western Blot, Control, Cell Culture

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) (B) RIF1-BRCA1 co-IP analysis. Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. FLAG IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. (C) RIF1-BRCA1 co-IP analysis. Reciprocal co-IP to (A)(B). Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. GFP IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. (D) RIF1-BRCA1-BRCT co-IP analysis. Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with mCherry-BRCA1-BRCT plasmid. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging. (E) Schematic representation of RIF1 constructs used in (D) and their co-IP analysis outcomes with BRCA1-BRCT. (F) Western blot analysis of RIF1-L-Phospho-S2265 signal in HeLa RIF1 KO cells supplemented with Dox-inducible RIF1-L or RIF1-L-pp1bs. (HeLa cell characterisation see Fig.S3E). (G) Representative images of RIF1-BRCA1 PLA foci in HeLa RIF1 KO cells (left) and in RIF1 KO cells supplemented with RIF1-L (middle) or RIF1-L-pp1bs (right). HU: 4mM 24hr. Scalebar: 10µm. (H) Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as (G). Experiment was repeated three times (see also Fig.S5G), and one representative result is shown. (I) RIF1-BRCA1-BRCT co-IP analysis. Flp-In TREx 293 cells expressing GFP-RIF1-L-pp1bs or GFP-RIF1-L were transfected with mCherry-BRCA1-BRCT plasmid. 16hr after transfection, cells were further treated with no, or 4h, or 24h of 4mM HU. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Co-Immunoprecipitation Assay, Expressing, Construct, Transfection, Plasmid Preparation, Staining, Imaging, Western Blot

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Representative images of GFP-RIF1-L or GFP-RIF1-L-pp1bs signal in HeLa cells, with indicated treatments, captured by fluorescence microscopy. unt: not treated with HU; HU: 4mM, 24hr. Scalebar: 10µm. (B) Quantification of GFP-RIF1-L or GFP-RIF1-L-pp1bs foci in cells from the experiment as (A). Means and standard errors of three independent experiments are plotted. p values calculated by two-tailed Student’s t test. (C) Left: Representative images of GFP-RIF1 fluorescence and BRCA1 immunofluorescence in HeLa RIF1 KO, +GFP-RIF1-L, and +GFP-RIF1-L-pp1bs cells. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. Right: BRCA1 nuclear signal intensity fold change. More than 200 nuclei were measured for each sample (see Fig.S6B for one representative experiment). Median intensity values from three independent experiments were recorded. Fold changes were determined by normalising to values of the RIF1 KO sample. Means and standard errors were shown. p values calculated by one-sample t test. (D) Left: Representative images of BRCA1 immunofluorescence in HeLa cells treated with siCtrl or siRIF1. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. Right: BRCA1 nuclear signal intensity fold change (normalised to siCtrl cells). Data plotted as described in (C). p values calculated by one-sample t test. (E) An example of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and γH2AX in HeLa cells. This sample was treated with 4mM 24hr HU. BRCA1 and γH2AX signals were generated by immunostaining. Scalebar: 5µm. (F) Quantification of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and γH2AX in cells from the experiment as (E). Means and standard errors of four independent experiments are plotted. (G) An example of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and RAD51 in HeLa cells. This sample was treated with 4mM 24hr HU. BRCA1 and RAD51 signals were generated by immunostaining. Scalebar: 5µm. (H) Quantification of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and RAD51 in cells from the experiment as (G). Means and standard errors of three independent experiments are plotted.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Fluorescence, Microscopy, Two Tailed Test, Immunofluorescence, Generated, Immunostaining

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Representative images of RAD51 immunofluorescence in HeLa cells that are RIF1 KO, or express indicated RIF1 constructs. (HeLa cell characterisation see Fig.S3E). Nuclei outlines were drawn in white. RAD51 signal is shown in orange. unt: not treated with HU; HU: 4mM 24hr. Scalebar: 10µm. (B) Percentage of nuclei containing indicated number of RAD51 foci in cells from the experiment as (A). Means and standard errors of three independent experiments were plotted. p values are calculated by chi-square tests. (C) Western blot analysis of BRCA1 depletion by siRNA in HeLa cells. (D) Representative images of RAD51 immunofluorescence in HeLa cells with indicated RIF1 expression and siRNA treatments. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. (E) Percentage of nuclei containing indicated number of RAD51 foci in cells from the experiment as (D). Means and standard errors of two independent experiments were plotted. p values are calculated by chi-square tests. (F) Schematic diagram of the reporter construct in HCT116 HR reporter cell lines to assess homologous recombination-mediated repair at Cas9n-induced broken forks. (G) Flow cytometry analysis of HR-mediated fork repair assessed by the reporter shown in (F), in HCT116 HR reporter cells expressing indicated RIF1 derivatives made at the endogenous RIF1 loci by CRISPR modification. (See Fig.S7G for HCT116 HR reporter cell characterisation). Means and standard errors of three independent experiments are plotted. p values are calculated by two-tailed paired Student’s t test. (H) Number of micronuclei per 100 cells in HeLa cells with indicated RIF1 expression and treatments. unt: not treated with HU; HU: 4mM 24hr. Means and standard errors of three independent experiments were plotted. p values are calculated by one-tailed Student’s t test.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Immunofluorescence, Construct, Western Blot, Expressing, Homologous Recombination, Flow Cytometry, CRISPR, Modification, Two Tailed Test, One-tailed Test

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Model of how RIF1-L promotes recovery from replication stress. (i) Replication fork progresses in unperturbed condition. (ii) Replication fork stalls upon replication stress. RIF1 and BRCA1 are independently recruited to stalled forks. (iii) Persistent stalling leads to fork breakage and subsequent formation of a single-ended DSB. RIF1-L interacts with BRCA1 dependent on phosphorylation of RIF-L S 2265 . (iv) RIF1-L-BRCA1 complex facilitates the loading of RAD51 onto seDSBs. RAD51 nucleofilament thereby initiate strand invasion into the sister chromatid and proceed to homology-directed repair. (B) A speculative model proposing that RIF1-L may bridge the broken daughter and parental DNAs. Unmethylated H4K20 is enriched on newly-replicated nascent chromatin (pale orange octagons), enabling BRCA1-BARD1 recruitment. Di-methylated H4K20 is enriched on un-replicated parental chromatin (grey octagons), favouring 53BP1. We speculate that RIF1-L may bind BRCA1 via its C-terminal phosphorylated SPKF motif, while interacting with 53BP1 via its N-terminal residues. In this manner, RIF1-L may facilitate the homology pairing between sister chromatids. In the RIF1-L protein, pale blue curve represents the IDR region; red bar represents the S 2265 PKF motif. ‘N’ and ‘C’ marks N-terminal and C-terminal of the RIF1-L protein.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Methylation

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: RIF1-L phospho-SPKF peptide binds to tandem BRCT domain of BRCA1 in vitro (A) (B) Fluorescence polarisation analysis of binding affinities between BRCA1-BRCT domain and RIF1-L phospho-peptide with indicated treatment or mutations. (C) Crystal structure of RIF1-L phospho-peptide (stick presentation) in complex with BRCA1-BRCT domain (space-filling presentation), solved by X-ray diffraction. The phosphorus atom is represented in orange. Residues on RIF1-L phospho-peptide are labelled with pink text (S2265,K2267,F2268,K2269). Residue on BRCA-BRCT domain is labelled with green text (E1698). (D) Model of RIF1-L interaction with BRCA1. In RIF1-L protein presentation, purple bards indicate PP1-interacting motifs; pink box indicates Exon 31; red bar indicates the ‘S 2265 PKF’ motif. RIF1-L phosphoS 2265 PKF motif binds to the C-terminal tandem BRCT domains of BRCA1.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: In Vitro, Fluorescence, Binding Assay, Residue

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: RIF1-L isoform exhibits proximity with BRCA1 in vivo under replication stress (A) Representative images of RIF1-BRCA1 PLA foci in RPE-1 cells with indicated treatments. Scalebar: 10µm. (B) Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as (A). In this and all following Tukey box-and-whisker plots, box represents 1st to 3rd quartile, horizontal line inside the box represents median. p values are calculated by Mann-Whitney test for non-parametric distributions. n.s. p >0.05; * p ≤0.05; ** p ≤0.01; *** p ≤0.001; **** p ≤0.0001. (C) RIF1-BRCA1 PLA analysis in RPE-1 cells with indicated treatments. HU: 4mM 24hr; APH: 4µM 24hr; CPT: 100nM 24hr. (D) Schematic representation of RIF1-L Morpholinos (bright blue lines) targeting the splicing signals for RIF1-L Exon31 inclusion. The RIF1-L morpholinos were designed to inhibit spliceosome recruitment leading to the skipping of Exon 31 during pre-mRNA splicing. Treatment with RIF1-L Morpholinos is expected to prevent the generation of RIF1-L mRNA. (E) Gel analysis of RT-PCR-based analysis to distinguish RIF1-L and RIF1-S transcripts in Control and RIF1-L Morpholino-treated RPE-1 cells. The upper band corresponds to RIF1-L mRNA and the lower band corresponds to RIF1-S mRNA. Experimental procedure described in Fig.S3A. (F) Western blot analysis of total RIF1 protein expression in Control and RIF1-L Morpholino-treated RPE-1 cells. Mo: abbreviation for Morpholino. (G) RIF1-BRCA1 PLA analysis in Control and RIF1-L Morpholino-treated RPE-1 cells with indicated HU treatments. Experiment was repeated twice (see also Fig.S3C), and one representative result is shown.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: In Vivo, Whisker Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A Western blot analysis of 53BP1 depletion in RPE-1 cells. (B) RIF1-BRCA1 PLA analysis in Control or 53BP1-depleted RPE-1 cells with indicated treatments. HU: 4mM 24hr. Experiment was repeated twice (see also Fig.S4A), and one representative result is shown. (C) Experiment procedure to label S phase cells with EdU, followed by RIF1-BRCA1 PLA analysis. An asynchronous RPE-1 cell culture was pulse labelled with EdU for 15 min, and then treated with no or 4mM HU for 4hr. Cells were fixed at the end of HU treatment and subjected to RIF1-BRCA1 PLA procedures. (D) Left: representative images acquired from the experiment described in (C). unt: not treated with HU; HU: 4mM 4hr. Scalebar: 10µm. Right: quantification of RIF1-BRCA1 PLA foci per nucleus. Experiment was repeated twice (see also Fig.S4D), and one representative result is shown. (E) RIF1-BRCA1 PLA analysis in RPE-1 cells collected at indicated timepoints after removal of HU. Experiment was repeated twice (see also Fig.S4F), and one representative result is shown. (F) RIF1-BRCA1 PLA analysis in RPE-1 cells following indicated kinase inhibitor and HU treatments. HU: 4mM 24hr; ATRi (VE-821): 1µM 24hr; Chk1i (PF-477736): 1µM 24hr. Experiment was repeated twice (see also Fig.S4G), and one representative result is shown.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Western Blot, Control, Cell Culture

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) (B) RIF1-BRCA1 co-IP analysis. Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. FLAG IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. (C) RIF1-BRCA1 co-IP analysis. Reciprocal co-IP to (A)(B). Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. GFP IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. (D) RIF1-BRCA1-BRCT co-IP analysis. Flp-In TREx 293 cells expressing indicated GFP-RIF1 constructs were transfected with mCherry-BRCA1-BRCT plasmid. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging. (E) Schematic representation of RIF1 constructs used in (D) and their co-IP analysis outcomes with BRCA1-BRCT. (F) Western blot analysis of RIF1-L-Phospho-S2265 signal in HeLa RIF1 KO cells supplemented with Dox-inducible RIF1-L or RIF1-L-pp1bs. (HeLa cell characterisation see Fig.S3E). (G) Representative images of RIF1-BRCA1 PLA foci in HeLa RIF1 KO cells (left) and in RIF1 KO cells supplemented with RIF1-L (middle) or RIF1-L-pp1bs (right). HU: 4mM 24hr. Scalebar: 10µm. (H) Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as (G). Experiment was repeated three times (see also Fig.S5G), and one representative result is shown. (I) RIF1-BRCA1-BRCT co-IP analysis. Flp-In TREx 293 cells expressing GFP-RIF1-L-pp1bs or GFP-RIF1-L were transfected with mCherry-BRCA1-BRCT plasmid. 16hr after transfection, cells were further treated with no, or 4h, or 24h of 4mM HU. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Co-Immunoprecipitation Assay, Expressing, Construct, Transfection, Plasmid Preparation, Staining, Imaging, Western Blot

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Representative images of GFP-RIF1-L or GFP-RIF1-L-pp1bs signal in HeLa cells, with indicated treatments, captured by fluorescence microscopy. unt: not treated with HU; HU: 4mM, 24hr. Scalebar: 10µm. (B) Quantification of GFP-RIF1-L or GFP-RIF1-L-pp1bs foci in cells from the experiment as (A). Means and standard errors of three independent experiments are plotted. p values calculated by two-tailed Student’s t test. (C) Left: Representative images of GFP-RIF1 fluorescence and BRCA1 immunofluorescence in HeLa RIF1 KO, +GFP-RIF1-L, and +GFP-RIF1-L-pp1bs cells. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. Right: BRCA1 nuclear signal intensity fold change. More than 200 nuclei were measured for each sample (see Fig.S6B for one representative experiment). Median intensity values from three independent experiments were recorded. Fold changes were determined by normalising to values of the RIF1 KO sample. Means and standard errors were shown. p values calculated by one-sample t test. (D) Left: Representative images of BRCA1 immunofluorescence in HeLa cells treated with siCtrl or siRIF1. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. Right: BRCA1 nuclear signal intensity fold change (normalised to siCtrl cells). Data plotted as described in (C). p values calculated by one-sample t test. (E) An example of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and γH2AX in HeLa cells. This sample was treated with 4mM 24hr HU. BRCA1 and γH2AX signals were generated by immunostaining. Scalebar: 5µm. (F) Quantification of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and γH2AX in cells from the experiment as (E). Means and standard errors of four independent experiments are plotted. (G) An example of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and RAD51 in HeLa cells. This sample was treated with 4mM 24hr HU. BRCA1 and RAD51 signals were generated by immunostaining. Scalebar: 5µm. (H) Quantification of co-localisation between GFP-RIF1-L-pp1bs, BRCA1 and RAD51 in cells from the experiment as (G). Means and standard errors of three independent experiments are plotted.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Fluorescence, Microscopy, Two Tailed Test, Immunofluorescence, Generated, Immunostaining

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Representative images of RAD51 immunofluorescence in HeLa cells that are RIF1 KO, or express indicated RIF1 constructs. (HeLa cell characterisation see Fig.S3E). Nuclei outlines were drawn in white. RAD51 signal is shown in orange. unt: not treated with HU; HU: 4mM 24hr. Scalebar: 10µm. (B) Percentage of nuclei containing indicated number of RAD51 foci in cells from the experiment as (A). Means and standard errors of three independent experiments were plotted. p values are calculated by chi-square tests. (C) Western blot analysis of BRCA1 depletion by siRNA in HeLa cells. (D) Representative images of RAD51 immunofluorescence in HeLa cells with indicated RIF1 expression and siRNA treatments. All samples were treated with 4mM 24hr HU. Scalebar: 10µm. (E) Percentage of nuclei containing indicated number of RAD51 foci in cells from the experiment as (D). Means and standard errors of two independent experiments were plotted. p values are calculated by chi-square tests. (F) Schematic diagram of the reporter construct in HCT116 HR reporter cell lines to assess homologous recombination-mediated repair at Cas9n-induced broken forks. (G) Flow cytometry analysis of HR-mediated fork repair assessed by the reporter shown in (F), in HCT116 HR reporter cells expressing indicated RIF1 derivatives made at the endogenous RIF1 loci by CRISPR modification. (See Fig.S7G for HCT116 HR reporter cell characterisation). Means and standard errors of three independent experiments are plotted. p values are calculated by two-tailed paired Student’s t test. (H) Number of micronuclei per 100 cells in HeLa cells with indicated RIF1 expression and treatments. unt: not treated with HU; HU: 4mM 24hr. Means and standard errors of three independent experiments were plotted. p values are calculated by one-tailed Student’s t test.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Immunofluorescence, Construct, Western Blot, Expressing, Homologous Recombination, Flow Cytometry, CRISPR, Modification, Two Tailed Test, One-tailed Test

Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: ( A ) Model of how RIF1-L promotes recovery from replication stress. (i) Replication fork progresses in unperturbed condition. (ii) Replication fork stalls upon replication stress. RIF1 and BRCA1 are independently recruited to stalled forks. (iii) Persistent stalling leads to fork breakage and subsequent formation of a single-ended DSB. RIF1-L interacts with BRCA1 dependent on phosphorylation of RIF-L S 2265 . (iv) RIF1-L-BRCA1 complex facilitates the loading of RAD51 onto seDSBs. RAD51 nucleofilament thereby initiate strand invasion into the sister chromatid and proceed to homology-directed repair. (B) A speculative model proposing that RIF1-L may bridge the broken daughter and parental DNAs. Unmethylated H4K20 is enriched on newly-replicated nascent chromatin (pale orange octagons), enabling BRCA1-BARD1 recruitment. Di-methylated H4K20 is enriched on un-replicated parental chromatin (grey octagons), favouring 53BP1. We speculate that RIF1-L may bind BRCA1 via its C-terminal phosphorylated SPKF motif, while interacting with 53BP1 via its N-terminal residues. In this manner, RIF1-L may facilitate the homology pairing between sister chromatids. In the RIF1-L protein, pale blue curve represents the IDR region; red bar represents the S 2265 PKF motif. ‘N’ and ‘C’ marks N-terminal and C-terminal of the RIF1-L protein.

Article Snippet: The FLAG-BRCA1 plasmid was obtained from Addgene (#52504, pDEST 3x Flag-pcDNA5-FRT/TO-BRCA1 ).

Techniques: Methylation