title: The dynamics of replication licensing in live Caenorhabditis elegans embryos.
abstract: Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Remi
authors.initials: R
authors.lastname: Sonneville
authors.forename: Matthieu
authors.initials: M
authors.lastname: Querenet
authors.forename: Ashley
authors.initials: A
authors.lastname: Craig
authors.forename: Anton
authors.initials: A
authors.lastname: Gartner
authors.forename: J Julian
authors.initials: JJ
authors.lastname: Blow
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D059008:Time-Lapse Imaging
chemical_list: C522445:CDC-6 protein, C elegans; D029742:Caenorhabditis elegans Proteins; C475683:Cdt-1 protein, C elegans; D018797:Cell Cycle Proteins; D002843:Chromatin; D051738:Origin Recognition Complex; D004247:DNA; D008025:Ligases