Specific Binding of Eukaryotic ORC to DNA Replication Origins Depends on Highly Conserved Basic Residues

Sci Rep. 2015 Oct 12;5:14929. doi: 10.1038/srep14929.

Abstract

In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arginine / chemistry
  • Arginine / metabolism
  • Conserved Sequence
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism
  • DNA Replication*
  • Lysine / chemistry
  • Lysine / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Origin Recognition Complex / chemistry*
  • Origin Recognition Complex / genetics
  • Origin Recognition Complex / metabolism
  • Protein Binding
  • Replication Origin*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • ORC1 protein, S cerevisiae
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Arginine
  • Lysine