Figure 4: PrimPol RBM-A is critical for RPA-binding in vivo.
From: Molecular basis for PrimPol recruitment to replication forks by RPA
(a) Schematic detailing the domain architecture of N-terminal FLAG-tagged PrimPol transfected into HEK-293 derivative cells (Flp-In T-Rex-293). The RBD (480–560) containing the RBM-A and B sites is shown below with the mutations forming the A-KO (D519R and F522A) and B-KO (D551R and I554A) highlighted. (b) Flp-In T-Rex-293 cells were transfected with wild-type and RBM-A and B mutated PrimPol. Expression was confirmed by addition of 10 ng ml−1 doxycycline (indicated by Dox ± on figure) for 24 h and subsequent western blotting. (c) Flp-In T-Rex-293 cells transfected with FLAG-tagged wild-type (WT) PrimPol were grown in the presence or absence of doxycycline (10 ng ml−1, 24 h), FLAG-PrimPol was immunoprecipitated from the soluble cell lysate using anti-FLAG antibody and western blotted for PrimPol (anti-FLAG) and RPA (anti-RPA2). The presence and absence of doxycycline is indicated by ± Dox, ‘In’ indicates the input, ‘E1’, ‘E2’ and ‘E3’, indicate elutions 1, 2 and 3, respectively. (d) Immunoprecipitation of FLAG-PrimPolA-KO (D519R/F522A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. (e) Immunoprecipitation of FLAG-PrimPolB-KO (D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. (f) Immunoprecipitation of FLAG-PrimPolA+B-KO (D519R/F522A and D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline.
