Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila Melanogaster

Mol Cell Biol. 2007 Apr;27(8):3143-53. doi: 10.1128/MCB.02382-06. Epub 2007 Feb 5.

Abstract

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Animals
  • Bromodeoxyuridine
  • Chromosomes / metabolism
  • DNA / biosynthesis
  • DNA / metabolism*
  • DNA Replication*
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / isolation & purification
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / cytology
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / isolation & purification
  • Mutant Proteins / metabolism
  • Origin Recognition Complex / chemistry
  • Origin Recognition Complex / isolation & purification
  • Origin Recognition Complex / metabolism*
  • Poly A / genetics
  • Protein Binding
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / metabolism
  • Replication Origin*
  • Salivary Glands / cytology
  • Salivary Glands / metabolism

Substances

  • Amino Acids
  • Drosophila Proteins
  • Mutant Proteins
  • ORC6 protein, Drosophila
  • Origin Recognition Complex
  • Recombinant Fusion Proteins
  • Poly A
  • DNA
  • Bromodeoxyuridine