Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication
Figure 2
Orc1 specifically interacts with Rb in vitro.
(A) GST pull-down experiment performed by incubating GST alone or GST fused to the N-terminus (aa. 1–400) or the C-terminus (aa. 379–928) of Rb, immobilized on gluthatione-agarose beads, with in vitro translated [35S]-labelled Orc1 or Orc2 proteins. The upper panel shows the quantification of the [35S]-labelled protein after in vitro binding. The amount of radioactivity bound to the beads is indicated as a percentage of the input material. The lower panel shows the autoradiography. The Input lanes contain the labelled proteins prior to binding. A representative experiment of at least 3 performed is shown. (B) GST pull-down experiment performed by incubating GST, GST-Rb (379–928) or GST-E2F1 fusion proteins on gluthatione-agarose beads with in vitro translated [35S]-labelled Orc1 or Orc2. The results are shown as in panel (A). (C) Binding to Orc1 is specific for Rb. The scheme on the left side shows the conserved A/B pocket domains in the three members of the RB family, which were used as GST fusion proteins. The figure on the right side, shows the result of a GST pull-down experiment performed with these proteins, and in vitro translated Orc1 and Orc2. “Rb” for short corresponds to the C-terminus of Rb (aa. 379–928). (D) Binding to Orc1 requires the C-terminal region of Rb. The scheme on the left side of the figure shows the Rb truncated or mutated proteins used for mapping the domains required for binding to Orc1 (AE, containing the A, B and C pockets; AB, A and B pockets only; SE, C pocket only; AE Cys706Phe, which does not bind LxCxE proteins). These proteins were obtained as GST fusions and used for the GST pull-down experiment shown on the right side of the panel. (E) Schematic representation of the main functional domains of the Orc1 protein. BAH, bromo-adjacent homology domain; HP1, HP1 binding domain; AAA, ATPase domain; HW, putative DNA binding site. The fragments of Orc1 subsequently tested by in vitro GST pull-down are indicated by the corresponding amino acids on the left side. (F) GST pull-down experiment performed with the Orc1 fragments indicated in (E), labelled by in vitro translation, and challenged to GST or GST-Rb proteins. “Rb” for short corresponds to the C-terminus of Rb (aa. 379–928). (G) Sequence alignment showing the conserved LxCxE motif found in the Orc1 subunit of human, rat, mouse and hamster (upper part), and GST pull-down experiment performed with the in vitro translated Orc1 LPGRK protein (mutated in the LxCxE motif of Orc1) and the GST-fusion proteins AE, AB and SE (lower part).
