52504 pdest 3x flagpcdna5 frt to brca110  (Addgene inc)

Journal: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A , B Fluorescence polarisation analysis of binding affinities between BRCA1-BRCT domain and RIF1-L phospho-peptide with indicated treatment or mutations. Source data are provided as a file. 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: Fluorescence, Binding Assay, Residue

Journal: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A Representative images of RIF1-BRCA1 PLA foci in RPE-1 cells with indicated treatments. Scalebar: 10 µm. B Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as ( A ). p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons using the ‘unt’ sample as the control group. C RIF1-BRCA1 PLA analysis in RPE-1 cells with indicated treatments. HU: 4 mM 24 h; APH: 4 µM 24 h; CPT: 100 nM 24 h. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons using the ‘unt’ sample as the control group. 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 Supplementary Fig. . 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. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. In the Tukey box-and-whisker plots of this and the following figures, box represents 1st to 3rd quartile of data points. Horizontal line inside the box represents median. Whisker extending from box represent 1.5x interquartile range. Individual dots represent outliers greater than the value at whisker bound. n numbers of samples are listed in Supplementary Data . Numbers of independent experimental repeats is stated in ‘Statistics and Reproducibility’ section. Source data are provided as a file.

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

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Whisker Assay

Journal: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A Western blot analysis of 53BP1 depletion by siRNA in RPE-1 cells. B RIF1-BRCA1 PLA analysis in Control or 53BP1-depleted RPE-1 cells with indicated treatments. HU: 4 mM 24 h. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. 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 4 mM HU for 4 h. 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: 4 mM 4 h. Scalebar: 10 µm. Right: quantification of RIF1-BRCA1 PLA foci per nucleus. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. E RIF1-BRCA1 PLA analysis in RPE-1 cells collected at indicated timepoints after removal of HU. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. F RIF1-BRCA1 PLA analysis in RPE-1 cells following indicated kinase inhibitor and HU treatments. HU: 4 mM 24 h; ATRi (VE-821): 1 µM 24 h; Chk1i (PF-477736): 1 µM 24 h. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups.

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: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A , B RIF1-BRCA1 co-IP analysis. Flp-In T-REx 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 T-REx 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 T-REx 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 presented in Supplementary Fig. ). 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: 4 mM 24 h. Scalebar: 10 µm. H Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as ( G ). p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. I RIF1-BRCA1-BRCT co-IP analysis. Flp-In T-REx 293 cells expressing GFP-RIF1-L-pp1bs or GFP-RIF1-L were transfected with mCherry-BRCA1-BRCT plasmid. 16 h after transfection, cells were further treated with no, or 4 h, or 24 h of 4 mM 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: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A Representative images of GFP-RIF1-L or GFP-RIF1-L-pp1bs signal in HeLa cells, with indicated treatments. unt: not treated with HU; HU: 4 mM, 24 h. Scalebar: 10 µm. 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 (two-tailed) calculated by 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 4 mM 24 h HU. Scalebar: 10 µm. Right: BRCA1 nuclear signal intensity fold change. Median intensity values from three independent experiments were recorded (see Supplementary Fig. for one representative experiment). Fold changes were determined by normalising to values of the RIF1 KO sample. Means and standard errors were shown. p values (two-tailed) calculated by one-sample t test. Source data are provided as a file. D Left: Representative images of BRCA1 immunofluorescence in HeLa cells treated with siCtrl or siRIF1. All samples were treated with 4 mM 24 h HU. Scalebar: 10 µm. Right: BRCA1 nuclear signal intensity fold change (normalised to siCtrl cells). Data plotted as described in ( C ). p values (two-tailed) 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 4 mM 24 h HU. BRCA1 and γH2AX signals were generated by immunostaining. Scalebar: 5 µm. 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 4 mM 24 h HU. BRCA1 and RAD51 signals were generated by immunostaining. Scalebar: 5 µm. 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: Two Tailed Test, Fluorescence, Immunofluorescence, Generated, Immunostaining

Journal: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

Figure Lengend Snippet: A Representative images of RAD51 immunofluorescence in HeLa cells that are RIF1 KO, or express indicated RIF1 constructs. Nuclei outlines are drawn in white. RAD51 signal is shown in orange. unt: not treated with HU; HU: 4 mM 24 h. Scalebar: 10 µm. 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 are plotted. p values calculated by chi-square tests. Source data are provided as a Source Data file. 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 4 mM 24 h HU. Scalebar: 10 µm. 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 are plotted. p values calculated by chi-square tests. Source data are provided as a file. 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 Supplementary Fig. for HCT116 HR reporter cell characterisation). Dots of the same colour represent data collected from the same experiment. Means and standard errors of three independent experiments are plotted. p values (two-tailed) calculated by 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: 4 mM 24 h. Means and standard errors of three independent experiments are plotted. p values (one-tailed) calculated by paired 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: Nature Communications

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

doi: 10.1038/s41467-025-60817-y

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 . RAD51 localises to broken forks. ( 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: Phospho-proteomics, Methylation