Fig. 7
From: Structural basis of meiotic telomere attachment to the nuclear envelope by MAJIN-TERB2-TERB1
TRF1 recruitment by MAJIN–TERB2–TERB1. a Schematic of the human TERB1 sequence, highlighting the TRF1/TERB2-interacting region and phosphorylation site T648, aligned with constructs TRFB (561–658), T2B (585–642) and TBM (642–658). b Size-exclusion chromatography elution profiles of MAJINCore+BP1-TERB2–TERB1TRFB (1–147, 1–220, 561–658), TRF1TRFH (62–268), and MAJINCore+BP1-TERB2–TERB1TRFB WT and T648E (TERB1) upon incubation with a stoichiometric amount of TRF1TRFH. c EMSA analysing the ability of MAJINCore-TERB2–TERB1TRFB to interact with linear double-stranded DNA (dsDNA). Gel images are representative of at least three replicate EMSAs. d Quantification of DNA-binding by MAJINCore-TERB2–TERB1TRFB (MTTCore, black), TRF1 (green) and TRF1TRFH (purple) through densitometry of EMSAs performed using 25 nM (per molecule) FAM-dsDNA. Plots and apparent KD values were determined by fitting data to the Hill equation; error bars indicate standard error, n = 3 EMSAs. Source data are provided as a Source Data file. e Electron microscopy analysis of MAJINCore-TERB2–TERB1TRFB alone and in complex with plasmid dsDNA. Scale bars, 100 nm. f EMSA of MAJINCore-TERB2–TERB1TRFB upon incubation with TRF1TRFH (top) and TRF1TRFH alone (bottom) with linear double-stranded DNA (dsDNA). g EMSA of MAJINCore-TERB2–TERB1T2B with linear dsDNA upon incubation with TRF1TRFH. h Quantification of DNA-binding by MAJINCore-TERB2–TERB1TRFB (MTTCore/TRFB, black), MAJINCore-TERB2–TERB1T2B (MTTCore/T2B, red) and MAJINCore+BP1-TERB2–TERB1TRFB (MTTCore+BP1/TRFB, blue) upon incubation with TRF1TRFH (at molar ratios indicated) through densitometry of EMSAs; error bars indicate standard error, n = 3 EMSAs. i EMSA of MAJINCore+BP1-TERB2–TERB1TRFB with linear dsDNA upon incubation with TRF1TRFH. j EMSA of TRF1 (full length) with linear dsDNA upon incubation with MAJINCore-TERB2–TERB1TRFB. f-j Gel images and plots are representative of at least three replicate EMSAs. k Amylose pulldown of TRF1TRFH using MBP-fusion MAJINΔTM-TERB2–TERB1TRFB (MBP-MTTΔTM) with or without pre-incubation with plasmid dsDNA (as indicated). The uncropped gel image is shown in Supplementary Fig. 12f.
