Cryo-EM structures of the origin recognition complex bound to the essential elements of ARS DNA

Abstract

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Despite the high conservation of ORC in protein sequence among eukaryotes, its selectivity for replicators varies from species to species. The underlying molecular mechanism for this divergence of ORC in origin selectivity remains poorly understood. We recently resolved a series of cryo-EM structures of the Saccharomyces cerevisiae ORC bound to various origin DNA sequences that contains the ARS consensus sequence (ACS) and the B1-2 elements. In the structures, the ORC encircles DNA through extensive interactions with both phosphate backbone and bases. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific insertion helix (IH) motif of Orc4 in the major groove. Interestingly, the Orc4-IH motif is only conserved among yeast species but completely absent in higher eukaryotes. Deletion of the IH motif from Orc4 significantly changes the binding profile of ORC across yeast genome, making it behave more like its counterparts in higher eukaryotes. However, replacement of the S. cerevisiae IH with the one from Kluyveromyces lactis enables the chimeric ORC binding to the K. lactis ACS containing DNA in the cerevisiae genome. These data indicate that Orc4-IH plays a unique role in determining origin selectivity in yeast species. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact DNA. Through a cooperative effort from Orc2, Orc5 and Orc6 subunits, the origin DNA is bent at the B1 site away from the ORC’s central DNA-binding channel and wraps onto Orc6. Cryo-EM structures of ORC bound to different ARSs shows that DNA bending induced by ORC is universal to all yeast origins. The role of DNA bending by ORC in MCM loading will be presented and discussed.