ORC and Cdc6p Interact and Determine the Frequency of Initiation of DNA Replication in the Genome

Cell. 1995 Jun 2;81(5):667-76. doi: 10.1016/0092-8674(95)90528-6.

Abstract

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle Proteins / metabolism*
  • Chromosomes, Fungal
  • Cloning, Molecular
  • DNA Replication*
  • DNA, Fungal / biosynthesis
  • DNA-Binding Proteins / metabolism*
  • Electrophoresis, Gel, Two-Dimensional
  • Genes, Lethal
  • Genome, Fungal
  • Mutation
  • Origin Recognition Complex
  • Protein Binding
  • Recombinant Fusion Proteins / metabolism
  • Replication Origin*
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins*
  • Selection, Genetic
  • Suppression, Genetic

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • ORC2 protein, S cerevisiae
  • ORC5 protein, S cerevisiae
  • Origin Recognition Complex
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins