Architecture of the Saccharomyces cerevisiae Origin Recognition Complex Bound to Origins of DNA Replication

The Origin Recognition Complex (ORC) is thought to be required for the initiation of DNA replication in all eukaryotes. In Saccharomyces cerevisiae, ORC is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G1. I have investigated the architecture of yeast ORC bound to origin DNA. Determination of DNA residues important for ORCorigin association indicated that ORC interacts preferentially with one strand of the ARS] origin. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA crosslinking studies suggested that recognition of origin sequences is mediated by two groups of ORC subunits making sequence-specific contacts with two distinct regions of the DNA. Thus, the DNA-binding surface of ORC is formed by the coordinate action of multiple subunits. Electron microscopy (EM) of ORC showed that the complex has a shape consistent with the structure determined by protein-DNA crosslinking. These EM studies found that ORC is an elongated molecule with three lobes. Estimates of the molecular mass of ORC using EM were consistent with this elongated complex containing one copy of each of the six ORC subunits. ORC bound origin DNA along its long axis and interacted with approximately 50 base pairs as predicted from DNase I protection assays of ORC bound to origin DNA.