Fig. 4: CST physically interacts with RAD51 and targets it to RPA-coated ssDNA.
From: Crosstalk between CST and RPA regulates RAD51 activity during replication stress
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.
