Journal: bioRxiv
Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses
doi: 10.1101/2025.01.22.634233
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-labelled 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 5mM) to inhibit electrostatic interactions. Scale bars, 10 µm. C) LLPS assays with purified, Alexa488-labelled BRCA1-BARD1, pS5pS7 mCherry-hCTD and Cy5-RNA. BRCA1-BARD1 (at 5 µM) was mixed with phosphorylated CTD (2.5 µM) or 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), Alexa488, and Cy5. Scale bars, 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, pS5pS7 GFP-(CTD) 26 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. Scale bars, 10 µm.
Article Snippet: For the preparation of the Cy5-labelled ITS1 RNA, 8 µg of the template DNA was mixed with ATP, UTP, and GTP (at 2mM), CTP (at 1mM), Cy5-labelled CTP (5- Propargylamino-CTP-Cy5, Jena Bioscience, at 0.05 mM), and T7 RNA polymerase (at 1 µM) in a buffer containing 0.1 M Tris-HCl, pH 8.1; 1% Triton X-100; 16 mM MgCl 2 ; 10 mM spermidine; and 50 mM DTT.
Techniques: In Vitro, Binding Assay, Purification
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