brca1  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: The interaction between the BRCA1-BARD1 complex and RNAPII is direct and mediated by the phosphorylated CTD of RNAPII and the BRCT domains of BRCA1-BARD1 . A , schematic representation of the domains of BRCA1 and BARD1. Positions of investigated binding variants, the tags, and cleavage sites are indicated. B , western blot analysis of pull-downs from HEK293 lysates. HEK293 cells were lysed, and the lysate was cleared by centrifugation. To the supernatant, FLAG-BRCA1-BARD1 was added, and the samples were incubated with α-FLAG beads. As a control, the HEK293 lysate with no added BRCA1-BARD1 was used. The proteins were eluted using 3xFLAG peptide and the samples were analyzed using western blots. BRCA1-BARD1 interacts with RNAPII via the CTD phosphorylated on Ser2 and Ser5, respectively. Uncropped blot and gel images are provided in . C , SDS-PAGE analysis of in vitro pull-down assay between GST-(CTD) 26 and the BRCA1-BARD1 complex. Purified BRCA1-BARD1 was incubated with phosphorylated and nonphosphorylated GST-(CTD) 26 bound to glutathione beads. The samples were centrifuged and the input, unbound (supernatant) and bound (pellet) fractions were analyzed using the SDS PAGE. BRCA1-BARD1 interacts directly with GST-pS2pS5-(CTD) 26 and GST-pS5pS7-(CTD) 26 in vitro . Uncropped gel images are provided in A . D , SDS-PAGE analysis of in vitro pull-down assay between GST-pS5pS7-(CTD) 26 and BRCA1 BRCT and BARD1 BRCT, respectively. Purified BRCA1 BRCT and BARD1 BRCT, respectively, were incubated with GST-pS5pS7-(CTD) 26 bound to glutathione beads. The samples were centrifuged and the input, unbound (supernatant) and bound (pellet) fractions were analyzed using the SDS PAGE. Substitutions in the phosphoserine binding site (BRCA1 S1655F,K1702M , BARD1 S575F,K619A ) abolish the binding. Uncropped gel images are provided in B . E , SDS-PAGE analysis of in vitro pull-down assay between GST-pS5pS7-(CTD) 26 and BRCA1-BARD1 variants BRCA1 w.t. -BARD1 w.t. (BRCA1-BARD1), BRCA1 2M -BARD1 w.t. (BRCA1 S1655F,K1702M -BARD1), BRCA1 w.t. -BARD1 2M (BRCA1-BARD1 S575F,K619A ), and BRCA1 2M -BARD1 2M (BRCA1 S1655F,K1702M - BARD1 S575F,K619A ), respectively. Purified BRCA1-BARD1 variants were incubated with GST-pS5pS7-(CTD) 26 bound to glutathione beads. The samples were centrifuged and the input, unbound (supernatant) and bound (pellet) fractions were analyzed using the SDS PAGE. The inactivation of single tandem BRCT repeat (either BRCA1 or BARD1 BRCT, respectively) lead to the reduction of binding. Uncropped gel images are provided in C . CTD, C-terminal domain; RNAPII, RNA polymerase II.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: Binding Assay, Western Blot, Centrifugation, Incubation, Control, SDS Page, In Vitro, Pull Down Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: The BRCT domains of the BRCA1–BARD1 complex exhibit differences in their binding kinetics to the phosphorylated CTD of RNAPII . A , sensorgrams obtained by biolayer interferometry (BLI) demonstrating interactions of BRCA1 BRCT and BARD1 BRCT with GST-pS5pS7-(CTD) 26 . BRCA1 BRCT and BARD1 BRCT interact with the GST-pS5pS7-(CTD) 26 via the canonical phosphoserine binding site. Substitutions in the phosphoserine binding site (BRCA1 BRCT S1655F,K1702M , BARD1 BRCT S575F,K619A ) abolish the binding. The sensorgrams represent the mean of three measurements for each concentration. The data were analyzed in Octet Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software. Sensorgrams for individual concentrations of BRCA1 BRCT S1655F, K1702M , BARD1 BRCT S575F, K619A can be found in A . B , sensorgrams obtained by biolayer interferometry (BLI) and their respective fits ( left ). Comparison of association ( k as ) and dissociation ( k dis ) kinetic constants, and equilibrium dissociation ( K D ) constants for GST-pS5pS7-(CTD) 26 and BRCA1 and BARD1 BRCT, respectively, obtained by biolayer interferometry ( right ). BARD1 BRCT associates with GST-pS5pS7-(CTD) 26 more dynamically than BRCA1 BRCT. The sensorgrams represent the mean of three measurements for each concentration. The association and dissociation constants and the coefficient of determination (R 2 ) indicating the appropriateness of the fit were calculated in Octet Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software. C , structural alignment of BRCA1 BRCT (PDB: 1JNX, teal ) and BARD1 BRCT (PDB: 2NTE, purple ) obtained in UCSF Chimera. D , comparison of the amino-acid composition of the hydrophobic pocket of BRCA1 BRCT (1JNX, teal ) and the residues present on the homologous positions in BARD1 BRCT (2NTE, purple ). Close up from ( C ). E , comparison of sensorgrams obtained by biolayer interferometry (BLI) and their respective fits ( top ) of GST-pS5pS7-(CTD) 26 binding to BRCA1 BRCT M1775H and BARD1 BRCT H686H . Kinetic parameters (association ( k as ), dissociation ( k dis ), and dissociation ( K D ) constants) ( bottom ). The sensorgrams represent the mean of three measurements for each concentration. The data were analyzed in Octet Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software. Sensorgrams for individual concentrations can be found in C . CTD, C-terminal domain; PDB, Protein Data Bank; RNAPII, RNA polymerase II.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: Binding Assay, Concentration Assay, Software, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: Structural characterization of the BRCA1 BRCT domain bound to pS5 CTD peptide . A , crystal structure of BRCA1 BRCT ( gray ) with bound pS5 CTD peptide ( yellow ), PDB ID: 9QPX. The data collection and refinement statistics is provided in . B , detail of the BRCA1 BRCT phospho-peptide binding site ( gray ) with bound pS5 CTD peptide ( yellow ). Close up from ( A ). C , detail of the phosphoserine binding site of BRCA1 BRCT ( gray ) with bound pS5 CTD peptide ( yellow ). The pS5 of the CTD peptide is depicted in purple , the amino-acid residues interacting with pS5 are depicted in light violet . The hydrogen bonds were displayed as pseudo bonds using the structural analysis tool Hydrogen bonds in UCSF ChimeraX and are depicted in turquoise . The relax distance tolerance was 1.0 Å and the relax angle tolerance was 20.0. D , detail of the aromatic amino-acid binding pocket of BRCA1 BRCT ( gray ) with bound pS5 CTD peptide ( yellow ). The Y1 of the CTD is depicted in navy blue , the interacting amino-acid residues in light blue . The hydrogen bonds ( left ) were displayed as pseudobonds using the structural analysis tool Hydrogen bonds in UCSF ChimeraX and are depicted in turquoise . The relax distance tolerance was 1.0 Å and the relax angle tolerance was 20.0. van der Waals hydrophobic and stacking interactions ( right ) were displayed as pseudobonds using the structural analysis tool in UCSF ChimeraX and are depicted in black . The interacting atoms were identified based on van der Waals overlap ≥ −0.4 Å. E , validation of the obtained structural model by fluorescence anisotropy measurement. The assays were performed between BRCA1 BRCT domain and the indicated ligands (at 25 nM). Anisotropy data were plotted as a function of protein concentration and fitted to a single-site saturation with nonspecific binding model using XMGrace. F , comparison of binding of different ligands to BRCA1 BRCT. Alignment of the structures was created using UCSF ChimeraX. BRCA1 BRCT (from the structure with pCTD, PDB ID: 9QPX) is depicted in gray , pCTD peptide in yellow , BACH1 phosphopeptide (PDB: 1T29) in red , CtIP phosphopeptide (PDB: 1Y98) in magenta , Abraxas singly phosphorylated peptide (PDB: 4Y2G) in turquoise , ATRIP phosphopeptide (PDB: 4IGH) in blue . pCTD, phosphorylated CTD; PDB, Protein Data Bank; pS5-CTD, phosphorylated on serine 5-CTD.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: Binding Assay, Biomarker Discovery, Fluorescence, Protein Concentration, Comparison, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: The BRCA1-BARD1 complex forms liquid-like condensates in vitro , which accommodate phosphorylated CTD domain of RNAPII and RNA . A , schematic representation of BRCA1 and BARD1 domains. Positions of investigated binding mutants are indicated. B , liquid-liquid phase separation (LLPS) assays with purified, Alexa488-labeled BRCA1-BARD1. BRCA1-BARD1 (at 1.25 μM, 2.5 μM, and 5 μM) was mixed with the crowding agent (10% dextran). Bar chart ( top ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1-BARD1. Statistical significance was determined by unpaired t test. A nested scatterplot ( middle ) represents quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1-BARD1, with median area determined per dataset. Statistical significance was determined by nested t test. Representative images from three experiments ( bottom ) are depicted as an overlay of differential interference contrast (DIC) and GFP. Where indicated, hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions or ATP (at 5 mM) to inhibit electrostatic interactions. The scale bar represents 10 μm. C , LLPS assays with purified, Alexa488-labeled BRCA1-BARD1, mCherry-p-hCTD and Cy5-RNA. BRCA1-BARD1 (at 5 μM) was mixed with phosphorylated CTD (2.5 μM), and Cy5-ITS1 RNA (at 15 nM), respectively, in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), Alexa488, and Cy5. The scale bar represents 10 μm. D , bar chart ( top ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with the BRCA1-BARD1 complex shown in ( C ). Statistical significance was determined by unpaired t test. A nested scatterplot ( bottom ) represents quantification (n = 3) of an area of individual droplets from three independent experiments with the BRCA1-BARD1 complex shown in ( C ), with median area determined per dataset. Statistical significance was determined by nested t test. E , LLPS assays with purified, BRCA1-BARD1, mGFP-p-hCTD, and Cy5-RNA. BRCA1-BARD1 (at 5 μM) was mixed with phosphorylated CTD (2.5 μM) and Cy5-ITS1 RNA (at 15 nM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), GFP, and Cy5. The scale bar represents 10 μm. Quantification of the data can be found in ( B ). CTD, C-terminal domain; RNAPII, RNA polymerase II.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: In Vitro, Binding Assay, Purification, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: The BRCT repeats of the BRCA1-BARD1 form liquid-like condensates in vitro , which accommodate phosphorylated CTD domain of RNAPII and RNA . A , LLPS assays with mCherry-p-hCTD and Alexa488-labeled BRCA1 BRCT ( top ) and BARD1 BRCT ( bottom ). The BRCA 1 BRCT and BARD1 BRCT (present at the 2.4:1 protein to its CTD binding site ratio), respectively, were mixed with the crowding agent (10% dextran) in absence or presence of phosphorylated CTD (2.5 μM). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) with the signal for Alexa488 or mCherry. The scale bar represents 10 μm. B , bar chart ( top ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1 and BARD1 BRCT shown in ( A ). Statistical significance was determined by unpaired t test. A nested scatterplot ( middle ) represents quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1-BARD1 shown in ( A ), with median area determined per dataset. Statistical significance was determined by nested t test. Bar chart ( bottom ) depicts the proportion of droplets containing signals for Alexa488-labeled BRCT ( green ) and mCherry-p-hCTD ( red ). C , LLPS assays with purified BRCA1 BRCT and mGFP-p-hCTD. BRCA1 BRCT w.t. and BRCA1 BRCT S1655F,K1702M (present at the 2.4:1 protein to its CTD binding site ratio), respectively, were mixed with phosphorylated CTD (2.5 μM) in the absence or presence of a crowding agent (10% dextran). Representative images ( top ) from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions. The scale bar represents 10 μm. Bar chart ( middle ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments. Statistical significance was determined by unpaired t test. A nested scatterplot ( bottom ) represents quantification (n = 3) of an area of individual droplets from three independent experiments, with median area determined per dataset. Statistical significance was determined by nested t test. Representative images from the experiments with three individual BRCT concentrations can be found in A . D , LLPS assays with purified BARD1 BRCT and mGFP-p-hCTD. BARD1 BRCT w.t. and BARD1 BRCT S575F,K619A (present at the 2.4:1 protein to its CTD binding site ratio), respectively, were mixed with phosphorylated CTD (2.5 μM) in the absence or presence of a crowding agent (10% dextran). Representative images ( top ) from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions. The scale bar represents 10 μm. Bar chart ( middle ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments. Statistical significance was determined by unpaired t test. A nested scatterplot ( bottom ) represents quantification (n = 3) of an area of individual droplets from three independent experiments, with median area determined per dataset. Statistical significance was determined by nested t test. Representative images from the experiments with three individual BRCT concentrations can be found in C . CTD, C-terminal domain; LLPS, liquid-liquid phase separation; RNAPII, RNA polymerase II.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: In Vitro, Labeling, Binding Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: Characterization of disease-associated variants within the BRCT repeats of the BRCA1-BARD1 complex on their ability to promote condensation in vitro . A , positions of the investigated substitutions in BRCT domains. The crystal structure of BRCA1 BRCT with the pS5 CTD ligand ( left ) and the AlphaFold 3-generated model of the BARD1 BRCT with the pS5 CTD ligand ( right ) were used. Investigated substitutions are not located near the phospho-peptide binding sites and therefore should not interfere with the binding. B LLPS assays with purified BRCA1 BRCT and mGFP-p-hCTD. BRCA1 BRCT w.t., BRCA1 BRCT E1682K , and BRCA1 BRCT E1754K , respectively, present at the indicated protein to its CTD binding site ratios, were mixed with phosphorylated CTD (2.5 μM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. The scale bar represents 10 μm. C , bar chart ( top ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1 BRCT w.t., BRCA1 BRCT E1682K , and BRCA1 BRCT E1754K , respectively, present at the 2.4:1 protein to its CTD binding site ratio, and mGFP-p-hCTD, shown in ( B ), using the green fluorescent signal. Statistical significance was determined by unpaired t test. A nested scatterplot ( bottom ) representing quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1 BRCT, mGFP-p-hCTD, shown in ( B ), with median area determined per dataset. Statistical significance was determined by nested t test. Quantification of the experiments with four individual BRCT concentrations can be found in A . D , LLPS assays with purified BARD1 BRCT and mGFP-p-hCTD. BARD1 BRCT w.t., BARD1 BRCT E587K , BARD1 BRCT E665K , BARD1 BRCT S711R , and BARD1 BRCT K754N , respectively, present at the indicated protein to CTD binding site ratio, were mixed with phosphorylated CTD (2.5 μM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. The scale bar represents 10 μm. E , bar chart ( top ) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BARD1 BRCT w.t., BARD1 BRCT E587K , BARD1 BRCT E665K , BARD1 BRCT S711R , and BARD1 BRCT K754N , respectively, present at the 2.4:1 protein to its CTD binding site ratio, and mGFP-p-hCTD, shown in ( D ), using the green fluorescent signal. The analysis and visualization were performed as in ( C ). Quantification of the experiments with three BRCT concentrations can be found in B . CTD, C-terminal domain; LLPS, liquid-liquid phase separation; pS5-CTD, phosphorylated on serine 5-CTD.

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: In Vitro, Generated, Binding Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1–BARD1 complex

doi: 10.1016/j.jbc.2025.111010

Figure Lengend Snippet: Schematic model of the interaction between RNAPII and the BRCA1-BARD1 complex .The BRCA1-BARD1 complex may associate with the transcriptionally engaged RNAPII phosphorylated on Ser5 of the CTD (pCTD) via the BRCT domains of BRCA1 and BARD1 e . g ., during unscheduled pausing of RNAPII for instance at a double-stranded DNA break. The more stable interaction between the BRCA1 BRCT domain and the pCTD might serve to anchor the complex to RNAPII, whilst the BARD1 BRCT domain may engage more dynamically, thereby enabling the complex to sample the vicinity of the paused RNAPII. These interactions might contribute to the formation of condensates containing the BRCA1-BARD1 complex alongside the transcriptionally engaged RNAPII, although this remains to be confirmed in vivo . RNAPII, RNA polymerase II; CTD, C-terminal domain

Article Snippet: To express the DNA binding region of BRCA1 (amino acids 421–1079), the fragment of DNA, containing the coding sequence was amplified by PCR and cloned into 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning, addgene #55220).

Techniques: In Vivo

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