Fig. 2: DNA-binding characteristics of the CST and RPA complexes.
From: Crosstalk between CST and RPA regulates RAD51 activity during replication stress
a Schematic showing the design of our single-molecule FRET (smFRET) experiment to determine DNA-binding affinities. b Measurement of the DNA-binding affinity (Kd) of CST and RPA in ionic strengths of 50 mM and 150 mM KCl. The curve was fitted by means of a Hill slope equation in GraphPad Prism. At 50 mM KCl, the Kd values of RPA and CST are 0.12 nM (Hill slope = 3.14) and 0.12 nM (Hill slope = 2.74), respectively. At 150 mM KCl, the Kd values of RPA and CST are 0.11 nM (Hill slope = 2.83) and 0.29 nM (Hill slope = 1.4), respectively. Data points of each protein concentration represent mean ± S.D. calculated from three independent experiments. c (i) Illustration of our ssDNA pulldown assay. Excessive RPA was preincubated with a biotinylated 80-nt ssDNA linked to magnetic streptavidin beads. After adding CST, the ssDNA and its associated proteins were captured using a magnetic bead separator. (ii) RPA was preincubated with magnetic ssDNA beads and then the indicated amounts of CST were added under the condition of 50 mM KCl. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. (iii) Quantitative plot of amounts of RPA32 and STN1 in the bound fraction. Data represent mean ± S.D. calculated from three independent experiments. NS, not significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analyses were performed by one-way ANOVA with Tukey’s post hoc test. d Affintiy pulldown assay. Flag-CTC1-STN1-TEN1-His6 (1 μM) was incubated with RPA (1 μM) in the absence of DNA or in the presence of 30-nt ssDNA, followed by incubation with His-Tag Dynabeads to capture CST and associated proteins using a magnetic bead separator. The supernatant (S) and eluate (E) were analyzed. RPA alone is shown as a control. N = 3 biologically independent experiments. e CST physically associates with RPA in a DNA-dependent manner. FLAG-CTC1, Myc-STN1, and HA-TEN1 were co-expressed in HEK293T cells and then treated with 2 mM HU for 20 h. Cell lysates were treated with or without benzonase prior to immunoprecipitation with anti-Myc. Top: Western blot. Bottom: Agarose gel analysis of DNA removal after benzonase treatment. N = 3 biologically independent experiments.
