The dynamics of replication licensing in live Caenorhabditis elegans embryos

J Cell Biol. 2012 Jan 23;196(2):233-46. doi: 10.1083/jcb.201110080. Epub 2012 Jan 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / embryology*
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Division
  • Chromatin / metabolism
  • DNA / metabolism
  • DNA Replication*
  • Embryo, Nonmammalian / metabolism*
  • Ligases / metabolism
  • Origin Recognition Complex / metabolism
  • Time-Lapse Imaging

Substances

  • CDC-6 protein, C elegans
  • Caenorhabditis elegans Proteins
  • Cdt-1 protein, C elegans
  • Cell Cycle Proteins
  • Chromatin
  • Origin Recognition Complex
  • DNA
  • Ligases