The dynamics of replication licensing in live Caenorhabditis elegans embryos

Remi Sonneville, Matthieu Querenet, Ashley Craig, Anton Gartner (Lead / Corresponding author), J. Julian Blow (Lead / Corresponding author)

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    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.

    Original languageEnglish
    Pages (from-to)233-246
    Number of pages14
    JournalJournal of Cell Biology
    Volume196
    Issue number2
    DOIs
    Publication statusPublished - 23 Jan 2012

    Keywords

    • ORIGIN RECOGNITION COMPLEX
    • EUKARYOTIC DNA-REPLICATION
    • C-ELEGANS
    • CELL-CYCLE
    • MCM2-7 HELICASE
    • GEMININ HOMOLOG
    • ATP-HYDROLYSIS
    • RE-REPLICATION
    • S-PHASE
    • CHROMATIN

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