title: Orc2 protects ORCA from ubiquitin-mediated degradation.
abstract: Origin recognition complex (ORC) is highly dynamic, with several ORC subunits getting posttranslationally modified by phosphorylation or ubiquitination in a cell cycle-dependent manner. We have previously demonstrated that a WD repeat containing protein ORC-associated (ORCA/LRWD1) stabilizes the ORC on chromatin and facilitates pre-RC assembly. Further, ORCA levels are cell cycle-regulated, with highest levels during G(1), and progressively decreasing during S phase, but the mechanism remains to be elucidated. We now demonstrate that ORCA is polyubiquitinated in vivo, with elevated ubiquitination observed at the G(1)/S boundary. ORCA utilizes lysine-48 (K48) ubiquitin linkage, suggesting that ORCA ubiquitination mediates its regulated degradation. Ubiquitinated ORCA is re-localized in the form of nuclear aggregates and is predominantly associated with chromatin. We demonstrate that ORCA associates with the E3 ubiquitin ligase Cul4A-Ddb1. ORCA is ubiquitinated at the WD40 repeat domain, a region that is also recognized by Orc2. Furthermore, Orc2 associates only with the non-ubiquitinated form of ORCA, and Orc2 depletion results in the proteasome-mediated destabilization of ORCA. Based on the results, we suggest that Orc2 protects ORCA from ubiquitin-mediated degradation in vivo.
authors.affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Shen
authors.forename: Supriya G
authors.initials: SG
authors.lastname: Prasanth
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D016196:S Phase; D000071456:Sequestosome-1 Protein; D012995:Solubility; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C114234:CUL4A protein, human; D002843:Chromatin; D044842:Cullin Proteins; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D008239:Lysine