An Orc6 tether mediates ORC binding site switching during replication origin licensing

Abstract

During origin licensing, the origin recognition complex (ORC) loads two Mcm2-7 helicases onto DNA in a head-to-head conformation, establishing the foundation for subsequent bidirectional replication. Single-molecule experiments support a helicase-loading model in which one ORC loads both Mcm2-7 helicases at origins. For this to occur, ORC must release from its initial Mcm2-7 and DNA binding sites, flip over the helicase, and bind the opposite end of the Mcm2-7 complex and adjacent DNA to form the MO complex. Importantly, this binding-site transition occurs without ORC releasing into solution. Using a single-molecule FRET assay, we show that the N-terminal half of Orc6 tethers ORC to the N-terminal tier of Mcm2-7 (Mcm2-7N) during ORCs binding-site transition. This interaction involves both the folded Orc6 N-terminal domain (Orc6N) and the adjacent unstructured linker and forms before ORC releases from its initial Mcm2-7 interaction. The absence of this interaction increases the rate of ORC release into solution, consistent with a tethering function. CDK phosphorylation of ORC inhibits the tethering interaction, providing a mechanism for the known CDK inhibition of MO complex formation. Interestingly, we identify mutations in the Orc6 linker region that support MO complex formation but prevent double-hexamer formation by inhibiting stable second Mcm2-7 recruitment. Our study provides a molecular explanation for a one-ORC mechanism of helicase loading and demonstrates that Orc6 is involved in multiple stages of origin licensing. Significance StatementBidirectional DNA replication is critical for accurate and complete duplication of the genome. Eukaryotic organisms coordinate this through loading of two oppositely-oriented Mcm2-7 replicative helicases at origins of replication. Using single-molecule biochemical studies, we identified and characterized a tethering interaction during helicase loading that enables the helicase loader ORC (origin recognition complex) to flip between two Mcm2-7 and DNA binding sites to load the second helicases in the opposite orientation. This interaction is cell-cycle regulated as part of the mechanisms ensuring replication from a given origin initiates only once. Our findings have important implications for the multiple mechanisms of helicase loading and illustrate how single-molecule studies can complement structural studies to provide a full view of complex molecular assembly events.