DOI: 10.5061/dryad.547d7wm8z
title: A helicase-tethered ORC flip enables bidirectional helicase loading

description: 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.
description: 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
 (<i>E</i><sub>FRET</sub> 0.7-0.8), we noted the
 earliest time the &gt; 635 nm emission FRET signal rose above
 background noise level while a donor fluorophore was present (as
 determined by monitoring the signal in the &gt; 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. 
description: 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). <br> * Donor excited acceptor emission traces are
 named Gex_Rem (green excited, red emission).<br> * 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:  <br> &gt;&gt;[fn fp] =
 uigetfile <br> % use the dialog box to mouse click on the
 appropriate *.dat file, then type: <br>
 &gt;&gt;eval(['load ' [fp fn] '
 -mat']) <br> % 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.  <br> Column 1: aoinumber
 (between 1-500 depending on the experiment)<br> Column 2:
 framenumber (1-600, frames are acquired every 2.4s)<br> Column 3:
 amplitude (of fluorescence emission, integrated in a 5um pixel)<br>
 Column 4: xcenter of the aoi <br> 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.
  <br>