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.

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 (E_FRET 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. 

Source data is organized by figures in the main text (Figs 1-6). Each folder contains an excel sheet with DNA spots or Areas of Interest (AOIs) from the field of view that are used for analysis. Data for supplementary figures associated with the figures in the main text can be found within the individual figure folders.

We have provided integrated traces for the single-molecule experiments as .dat trace files that can be read and viewed in Matlab or the Matlab program imscroll, which is publicly available: https://github.com/gelles-brandeis/CoSMoS_Analysis (see instructions below). These integrated traces contain information on the integrated amplitude of fluorescence emission for 600 consecutive frames of acquisition at each AOI. 

The integrated trace files are named with as mmddyy_filenumber_Xex_Yem_traces.dat. The following three .dat files are generated for each experiment: 

* Donor excited donor emission traces are named Gex_Gem (green excited, green emission). 
* Donor excited acceptor emission traces are named Gex_Rem (green excited, red emission).
* Acceptor excited acceptor emission traces are named Rex_Rem (red excited, red emission).

To read the .dat integrated trace files in Matlab the user may type:  
>>[fn fp] = uigetfile 
% use the dialog box to mouse click on the appropriate *.dat file, then type: 
>>eval(['load ' [fp fn] ' -mat']) 
% This loads an aoifits structure into the Matlab command environment

The ‘aoifits’ structure allows access to the integrated trace data in the aoifits.data matrix member and a description of the columns of that matrix in the aoifits.dataDescription alphanumeric member. 

The first five columns within aoifits.data are the most relevant to view fluorescence emission intensities over time at individual AOIs.  
Column 1: aoinumber (between 1-500 depending on the experiment)
Column 2: framenumber (1-600, frames are acquired every 2.4s)
Column 3: amplitude (of fluorescence emission, integrated in a 5um pixel)
Column 4: xcenter of the aoi 
Column 5: ycenter of the aoi 

To determine which AOIs were used for analysis in each experiment, please see the excel sheet in each figure folder.