Structure and mutagenesis studies of the C-terminal region of licensing factor Cdt1 enable the identification of key residues for binding to replicative helicase Mcm proteins

J Biol Chem. 2010 May 21;285(21):15931-40. doi: 10.1074/jbc.M109.075333. Epub 2010 Mar 24.

Abstract

In eukaryotes, DNA replication is fired once in a single cell cycle before cell division starts to maintain stability of the genome. This event is tightly controlled by a series of proteins. Cdt1 is one of the licensing factors and is involved in recruiting replicative DNA helicase Mcm2-7 proteins into the pre-replicative complex together with Cdc6. In Cdt1, the C-terminal region serves as a binding site for Mcm2-7 proteins, although the details of these interactions remain largely unknown. Here, we report the structure of the region and the key residues for binding to Mcm proteins. We determined the solution structure of the C-terminal fragment, residues 450-557, of mouse Cdt1 by NMR. The structure consists of a winged-helix domain and shows unexpected similarity to those of the C-terminal domain of Cdc6 and the central fragment of Cdt1, thereby implying functional and evolutionary relationships. Structure-based mutagenesis and an in vitro binding assay enabled us to pinpoint the region that interacts with Mcm proteins. Moreover, by performing in vitro binding and budding yeast viability experiments, we showed that approximately 45 residues located in the N-terminal direction of the structural region are equally crucial for recognizing Mcm proteins. Our data suggest the possibility that winged-helix domain plays a role as a common module to interact with replicative helicase in the DNA replication-licensing process.

MeSH terms

  • Animals
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Helicases / chemistry
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • MADS Domain Proteins / chemistry
  • MADS Domain Proteins / genetics
  • MADS Domain Proteins / metabolism*
  • Mice
  • Mutagenesis
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Structure-Activity Relationship

Substances

  • CDC6 protein, mouse
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • MADS Domain Proteins
  • Nuclear Proteins
  • Ris2 protein, mouse
  • DNA Helicases

Associated data

  • PDB/2RQQ