A helicase-tethered ORC flip enables bidirectional helicase loading

Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Methods
CoSMoS data sets were collected and analyzed as described previously (Ticau et. al. 2015). By alternating between laser excitation wavelengths, we monitored the co-localization of both the donor fluorophore on ORC and acceptor fluorophore(s) on Mcm2-7 and/or Cdt1 with origin-DNA molecules. Double-hexamer formation events with no labeled ORC (donor) were excluded from data analysis. To determine the time of formation of the high-FRET state (EFRET 0.7-0.8), we noted the earliest time the > 635 nm emission FRET signal rose above background noise level while a donor fluorophore was present (as determined by monitoring the signal in the > 635 nm field when only the 532 nm laser was turned on). Only spots where the arrival of the acceptor fluorophore could be confirmed within 4.8 s were considered genuine instances of high FRET.