Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase

Mol Cell. 2006 Jan 6;21(1):29-39. doi: 10.1016/j.molcel.2005.11.023.

Abstract

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism*
  • Affinity Labels / metabolism
  • Cell Cycle / physiology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Chromosomal Proteins, Non-Histone
  • DNA Replication*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Minichromosome Maintenance Complex Component 7
  • Models, Biological
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Origin Recognition Complex / metabolism*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Affinity Labels
  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Fungal Proteins
  • Nuclear Proteins
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • TAH11 protein, S cerevisiae
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Triphosphate
  • MCM2 protein, S cerevisiae
  • MCM7 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 7