Fig 1: CST and RPA localize in close proximity on ssDNA in cells in response to replication stress.a Crosstalk among CST, RPA, and RAD51 on ssDNA subjected to replication stress. b CST lies in close proximity to RPA upon fork stalling. (i) PLA is a technique that detects the physical proximity of two different proteins. In principle, if the two proteins are <40 nm apart, fluorescence signal can be detected. In brief, the two target proteins are bound by specific primary antibodies. If the target proteins are sufficiently proximal, PLA secondary antibodies hosting oligonucleotides can be ligated by means of two PLUS/MINUS PLA oligos to circularize. The DNA polymerase phi29 then processes rolling-circle amplification, and the resulting copies can be detected by hybridizing the fluorescence-labeled oligonucleotide51. (ii), (iii) PLA assays were performed to establish the close proximity of CTC1/STN1 with RPA in HeLa cells treated with hydroxyurea (HU) for 3 h. Representative PLA images of CTC1/RPA32 (ii) and STN1/RPA32 (iii) are shown. Scatter plots from one experiment are shown here. Red lines represent mean values ± SEM. N: the number of cells analyzed in each sample. P values were calculated by one-way ANOVA. NS not significant, ***P < 0.001; ****P < 0.0001. c CST colocalizes with RPA on the same ssDNA in response to fork stalling. HeLa cells expressing Flag-CTC/Myc-STN1/HA-TEN1 were labeled with BrdU and treated with or without HU (3 h), followed by co-immunostaining with anti-Flag (red), anti-RPA32 pS33 (cyan), and anti-BrdU (green) antibodies. N = 3 biologically independent experiments.
Fig 2: CST physically interacts with RAD51 and targets it to RPA-coated ssDNA.a Affinity pulldown assay. Flag-CTC1-STN1-TEN1-His6 (1 µM) was incubated with RAD51 (1 µM), followed by incubation with His-Tag Dynabeads to capture the CST and associated proteins by means of a magnetic bead separator. The supernatant (S) and eluate (E) were analyzed by 15% SDS-PAGE with Coomassie blue staining. RAD51 alone is shown as a control. N = 3 biologically independent experiments. b (i) Schematic of the ssDNA pulldown experiment. (ii) Excessive RPA was preincubated with biotinylated 80-nt ssDNA linked to magnetic streptavidin beads. Upon addition of CST and RAD51, the ssDNA and its associated proteins were captured using a magnetic bead separator. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. (iii) Quantitative plot of amounts of RAD51 in the bound fraction. Data represent mean ± S.D. calculated from three independent experiments. c For ssDNA pulldown analysis, RPA was preincubated with magnetic ssDNA beads. Then CTC1?700N-ST and RAD51 were added to complete the reaction. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. N = 3 biologically independent experiments.
Fig 3: DNA-binding characteristics of the CST and RPA complexes.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.
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