ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci.
The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.
Cuvier
Olivier
O
Institute of Human Genetics, Centre National de la Recherche Scientifique, Genome Dynamics and Development, 141, Rue de la Cardonille, 34396 Montpellier, France.
Lutzmann
Malik
M
Méchali
Marcel
M
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology; D007399:Interphase / Q000502:physiology*; D008938:Mitosis / Q000502:physiology*; D051738:Origin Recognition Complex / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000502:physiology*
D002843:Chromatin; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; C097703:XORC2 protein, Xenopus; C102178:XOrc1 protein, Xenopus; D029867:Xenopus Proteins; D016203:CDC2 Protein Kinase