title: Ku80 binds to human replication origins prior to the assembly of the ORC complex.
abstract: The Ku heterodimer, an abundant nuclear protein, binds DNA replication origins in a sequence-specific manner and promotes initiation. In this study, using HCT116 Ku80+/- haplo-insufficient and Orc2(delta/-) hypomorphic cells, the order of binding of Ku and the human origin recognition complex (HsORC) was determined. The nuclear expression of Ku80 was found to be decreased by 60% in Ku80+/- cells, while its general association with chromatin was decreased by 33%. Coimmunoprecipitation studies indicated that the Ku heterodimer associates specifically with the human HsOrc-2, -3, -4, and -6 subunits. Chromatin immunoprecipitation (ChIP) experiments, using cells synchronized to late G1, showed that the association of Ku80 with the lamin B2, beta-globin, and c-myc origins in vivo was decreased by 1.5-, 2.3-, and 2.5-fold, respectively, in Ku80+/- cells. The association of HsOrc-3, -4, and -6 was consistently decreased in all three origins examined in Ku80+/- cells, while that of HsOrc-2 showed no significant variation, indicating that the HsOrc-3, -4, and -6 subunits bind to the origins after Ku80. In Orc2(delta/-) cells, the association of HsOrc-2 with the lamin B2, beta-globin, and c-myc origins was decreased by 2.8-, 4.9-, and 2.8-fold, respectively, relative to wild-type HCT116 cells. Furthermore, nascent strand abundance at these three origins was decreased by 4.5-, 2.3-, and 2.6-fold in Orc2(delta/-) relative to HCT116 cells, respectively. Interestingly, the association of Ku80 with these origins was not affected in this hypomorphic cell line, indicating that Ku and HsOrc-2 bind to origins independently of each other.
authors.affiliation: McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.
authors.forename: Sahar
authors.initials: S
authors.lastname: Sibani
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Price
authors.forename: Maria
authors.initials: M
authors.lastname: Zannis-Hadjopoulos
mesh_terms: D034961:Antigens, Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D051738:Origin Recognition Complex; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin; D017384:Sequence Deletion
chemical_list: D034961:Antigens, Nuclear; D002843:Chromatin; D004268:DNA-Binding Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D020033:Protein Isoforms; D021122:Protein Subunits; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen