FIG. 6.

TRF2 phosphomimetic mutant S20E abrogates RNA binding, ORC recruitment, and OriP DNA replication. (A) GST, GST-TRF2(1-90), GST-TRF2(1-90)(S20A), and GST-TRF2(1-90)(S20E) were assayed by EMSA for RNA binding to a G-rich probe, (UUAGGG)₈ (left), or a C-rich probe, (CCCUAA)₈ (right). (B) GST, GST-TRF2(1-90), GST-TRF2(1-90)(S20A), and GST-TRF2(1-90)(S20E) were incubated with HeLa nuclear extracts (0.5 ml at 5 mg/ml), washed extensively, and then assayed by SDS-PAGE for the recruitment of ORC2 (top) or control PCNA (middle). Loading controls for GST (bottom) or Coomassie staining is indicated below. Std, standard. (C) Transient DNA replication assays were performed with HCT116 cells with an OriP plasmid coexpressing the EBNA1 protein. Cells were cotransfected with a cytomegalovirus (CMV)-Flag vector or CMV-FLAG-TRF2(FL) or the S20A, S20E, or ΔB mutant and then assayed at 72 h posttransfection for OriP replication. Recovered plasmid DNA was digested with DpnI plus BamHI (top) or BamHI alone (bottom) and assayed by Southern blotting. (D) Quantification of at least three independent transfection assays represented in C. (E) Western blot analysis of FLAG-TRF2 proteins (anti-FLAG) transfected for the replication assays shown in C. EBNA1 and actin served as transfection and loading controls, respectively.