Cyclin E-CDK2 phosphorylation controls ORC1 and RB interaction. (A) Interaction between purified RB and ORC1 in a MBP pull down assay. MBP-fused wild-type RB was bound to amylose resin and further incubated with in vitro translated, S35-labeled wild type ORC1 or its mutants in the presence or absence of Cyclin E-CDK2 and 1 mM ATP. Beads were isolated and bound proteins were separated by gel electrophoresis. MBP was used as a control in the assay. (B) Alignment of ORC1 sequences is shown with conserved LxCxE motif. The conserved residues of LxCxE motif are indicated with different colors. The alignment shows conserved residues in ORC1 from different species in vertebrate and invertebrate classes (Invertebrates: Brugia malayi, Caenorhabditis briggsae, Caenorhabditis elegans, Strongylocentrotus purpuratus, Culex quinquefasciatus, Apis mellifera, Drosophila melanogaster, Aedes aegypti, and Pediculus humanus; Vertebrates: Danio rerio, Xenopus laevis, Xenopus tropicalis, Gallus gallus, Taeniopygia guttata, Mus musculus and Homo sapiens). (C) Interaction between ORC1 and RB in a MBP pull-down assay. GST-fused wild-type RB or its mutant proteins were incubated with wild type MBP-ORC1 in the presence or absence of Cyclin E-CDK2 and/or 1 mM ATP. Amylose-bead-bound proteins were isolated and bound proteins were separated by gel electrophoresis followed by immunoblotting with anti-GST antibody. Recombinant MBP was used as a control in the assay. ORC, Origin Recognition Complex; RB, Retinoblastoma; GST, Glutathione S transferase; MBP, Maltose binding protein.