Editor's evaluation: A helicase-tethered ORC flip enables bidirectional helicase loading

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 origin–recognition complex (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 single-molecule Förster resonance energy transfer (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. Editor's evaluation The initiation of DNA replication in eukaryotes is preceded by the assembly of a pre-Replicative Complex (pre-RC) at all potential origins of DNA replication during the G1 phase of the cell division cycle. The pre-RC contains a double hexamer of Mcm2-7 subunits and each hexamer eventually becomes the core of the two replicative helicases during the initiation of DNA synthesis. The current paper addresses the role of ORC and Cdc6 in loading the Cdt1-bound Mcm2-7 hexamer onto origin DNA and the data show that a single ORC molecule can load two Mcm2-7 hexamers in a sequential loading reaction that involves ORC flipping on the origin DNA. The results nicely complement other studies that show a detailed pathway for pre-RC assembly. https://doi.org/10.7554/eLife.74282.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The eukaryotic DNA replication machinery first assembles and initiates synthesis at DNA sites called origins of replication. During G1, all potential origins are licensed by loading two Mcm2-7 replicative DNA helicases around the origin DNA in an inactive, head-to-head fashion (Abid Ali et al., 2017; Evrin et al., 2009; Li et al., 2015; Remus et al., 2009). This conformation prepares the helicases to initiate bidirectional replication upon activation during S phase. The Mcm2-7 complex is the core enzyme of the eukaryotic replicative helicase and its loading onto DNA is restricted to G1 phase (Aparicio et al., 1997; Diffley et al., 1994). This constraint prevents helicases from being loaded onto replicated DNA, ensuring that no part of the genome is replicated more than once per cell cycle (Siddiqui et al., 2013). Interactions between three proteins and the Mcm2-7 helicase direct eukaryotic helicase loading (Bell and Labib, 2016). The origin–recognition complex (ORC) binds origin DNA and then recruits Cdc6 (Bell and Stillman, 1992; Speck et al., 2005). The resulting ORC–Cdc6 complex encircles the origin DNA (Feng et al., 2021; Schmidt and Bleichert, 2020). Mcm2-7 in complex with Cdt1 associates with ORC-Cdc6 and adjacent DNA to form the short-lived ORC–Cdc6–Cdt1–Mcm2-7 (OCCM) complex (Sun et al., 2013; Ticau et al., 2015). Loading of a second Cdt1-bound-Mcm2-7 hexamer, oriented in the opposite direction of the first, completes formation of the Mcm2-7 ‘double hexamer’. Multiple mechanisms have been proposed to explain how two oppositely oriented helicases are loaded at an origin. In addition to a primary ORC-binding site, natural origins include at least one additional weaker, inverted ORC-binding site (Chang et al., 2011; Palzkill and Newlon, 1988; Wilmes and Bell, 2002). These sequences can be located at a variety of distances from one another but are typically less than ~60 bp apart (Chang et al., 2011). One mechanism for helicase loading proposes that distinct ORC molecules bound at the inverted DNA sites recruit the two helicases. This model is supported by evidence that mutations in the Mcm3 C-terminus predicted to interfere with interactions between Mcm2-7 and the ORC–Cdc6 complex prevent recruitment of both the first and second helicases (Coster and Diffley, 2017; Frigola et al., 2013). In addition, for ensemble experiments two ORC DNA-binding sites are required for successful helicase loading in vitro and origin function in vivo (Coster and Diffley, 2017). In contrast, single-molecule helicase-loading experiments with the ARS1 origin showed that a single ORC molecule can direct loading of both Mcm2-7 helicases (Ticau et al., 2015). A single ORC model is also supported by time-resolved cryoelectron microscopy (cryo-EM) experiments showing predominantly one ORC molecule bound to the DNA in each helicase-loading intermediate observed on the ARS1 origin (Miller et al., 2019). A goal of the current studies is to address these apparently contradictory observations. Structural studies have revealed important intermediates in helicase loading. ORC has been shown to bend DNA prior to Mcm2-7 recruitment (Li et al., 2018). Cryo-EM studies of the OCCM intermediate show that the first Mcm2-7 hexamer interacts with and encircles the DNA adjacent to ORC–Cdc6 (Sun et al., 2013; Yuan et al., 2017). These and related structures also reveal that interaction of the C-terminal region of ORC with the C-terminal region of Mcm2-7 mediates recruitment of the first Mcm2-7 hexamer (Yuan et al., 2020). ATP hydrolysis is required to proceed beyond the OCCM, and structures of intermediates on-pathway to the Mcm2-7 double hexamer had been elusive until a recent time-resolved cryo-EM study (Miller et al., 2019). Intriguingly, this study observed a complex in which an inverted ORC is engaged with the N-terminal region of the first Mcm2-7. Although it was suggested that formation of the inverted complex might be required to load double hexamers, whether one or two ORC molecules were required to form the complex and load the two Mcm2-7 helicases remained unresolved. Further, this study could not directly examine how the inverted complex was integrated into the sequence of events in helicase loading. A second goal of the current studies is to ask if a single ORC protein can mediate formation of all these stable intermediates and to understand how transitions between the intermediates are coordinated during helicase loading. Single-molecule biochemical studies have provided kinetic and mechanistic insights into helicase loading that complement structural studies. Single-molecule studies demonstrated that the two Mcm2-7 complexes in each double-hexamer associate with origin DNA in a one-at-a-time manner (Ticau et al., 2015). Single-molecule FRET (sm-FRET) has been used to monitor opening and closing of the interface between Mcm2 and Mcm5 (Bochman and Schwacha, 2008; Samel et al., 2014) that provides DNA access to the central channel of the Mcm2-7 ring (Ticau et al., 2017). These studies revealed that Mcm2-7 is recruited with an open gate that closes around origin DNA substantially after initial DNA association. Such approaches also revealed that recruitment and loading of each Mcm2-7 involves a separate set of Cdc6 and Cdt1 molecules. Following each Mcm2-7 recruitment, Cdc6 and Cdt1 are released sequentially, and the Mcm2-7 ring closes concomitant with Cdt1 release (Ticau et al., 2015; Ticau et al., 2017). This connection between Cdt1 release and ring closing is consistent with structural data that suggest that Cdt1 holds the Mcm2-7 ring open at the Mcm2-5 gate (Frigola et al., 2017; Zhai et al., 2017). The mechanism that drives recruitment of head-to-head Mcm2-7 hexamers remains unclear (Bell and Labib, 2016; Lewis and Costa, 2020). Here, we generate data supporting a model that reconciles the apparently inconsistent observations regarding ORC and helicase loading and directly observe how a single ORC guides double-hexamer formation. We monitor ORC–Mcm2-7 interactions in real-time using sm-FRET and show that recruitment of each Mcm2-7 hexamer is accompanied by a short ‘OM’ interaction with the same ORC protein. Prior to recruiting the second Mcm2-7 hexamer, ORC forms a distinct intermediate (referred to as ‘MO’) with the initially loaded Mcm2-7. The transition between the OM and MO intermediates is rapid and requires Cdt1 release. Forming the MO intermediate allows ORC to release from its initial binding site and flip over the initially loaded Mcm2-7, positioning ORC to rebind DNA at an inverted binding site without release into solution. The resulting MO intermediate recruits the second Mcm2-7 and is only disrupted when Mcm2-7 double-hexamer formation is initiated. Our findings reveal a highly coordinated series of events that ensures two Mcm2-7 helicases are loaded as head-to-head pairs poised to initiate bidirectional replication. Results Monitoring ORC–Mcm2-7 interactions during helicase recruitment To investigate the dynamics of ORC–Mcm2-7 interactions during helicase loading, we developed a sm-FRET assay for the initial interaction between these proteins based on previously described single-molecule helicase-loading experiments (Ticau et al., 2015). Using the structure of the OCCM as a guide (Yuan et al., 2017), we modified ORC and Mcm2-7 at sites that are proximal (~35 Å apart) during recruitment of the first Mcm2-7 (Figure 1a). ORC was labeled with a donor fluorophore at the Orc5 C-terminus (ORC5C-549) and Mcm2-7 was labeled with an acceptor fluorophore at the Mcm2 C-terminus (Mcm2-72C-649). Importantly, the fluorescent labels did not interfere with protein function in ensemble helicase-loading assays (Figure 1—figure supplement 1). To monitor ORC–Mcm2-7 interactions during loading, purified ORC5C-549, Mcm2-72C-649, Cdt1, and Cdc6 were incubated with fluorescently labeled surface-tethered origin DNA. Using total internal reflection fluorescence microscopy, we monitored the colocalization of the fluorescently modified proteins with individual DNA molecules (Friedman and Gelles, 2015). Alternate excitation of the donor and acceptor fluorophores (Figure 1—figure supplement 2b) allowed observation of the association of each labeled protein with origin DNA, and determination of the apparent FRET efficiency (EFRET) during donor excitation measured ORC–Mcm2-7 C-terminal interactions (Figure 1a, b). We will refer to the ORC–Mcm2-7 interactions monitored using FRET between ORC5C-549 and Mcm2-72C-649 as ‘OM interactions’. Figure 1 with 4 supplements see all Download asset Open asset Origin–recognition complex (ORC) recruits two Mcm2-7 helicases through sequential OM interactions. (a) A schematic of the molecular events during helicase loading. The ORC and Mcm2-7 proteins are labeled at their C-termini (C) which form an interface during first Mcm2-7 recruitment (Yuan et al., 2017). ORC5C-549 is labeled with a donor fluorophore (D; green circles) and Mcm2-72C-649 is labeled with an acceptor fluorophore (A; red circles). The associated fluorescence images show single frames of ORC, Mcm2-7, and raw FRET fluorescence spots at a single DNA molecule in green, red, and blue outlines, respectively. We use gray and yellow coloring to indicate the first and second Mcm2-7 hexamers, respectively. (b) Schematic of DNA-bound ORC5C-549 and Mcm2-72C-649 proteins. When ORC5C-549 and Mcm2-72C-649 are not associated, excitation of the donor (Dex) only yields emission from the donor fluorophore (Dem). However, when ORC5C-549 and Mcm2-72C-649 are in proximity, we observe emission from the acceptor (Aem) on donor excitation due to FRET, and corresponding lower emission from the donor fluorophore (Dem). (c) A representative trace showing ORC and Mcm2-7 associations with DNA and ORC–Mcm2-7 (OM) interactions during helicase loading. Donor-excited fluorescence record shows ORC5C-549 association (green: Dex, Dem panel) and acceptor-excited fluorescence record shows Mcm2-72C-649 association (red: Aex, Aem panel). Gray arrows link to the corresponding molecular events shown in (a). Interaction between ORC and Mcm2-7 decreases the distance between the donor and acceptor fluorophores and results in increased apparent FRET efficiency (EFRET, blue: Dex, [Aem/ (Aem + Dem)] panel). EFRET values are calculated using donor-excited emission from the donor and acceptor fluorophores (see Materials and methods) and they are therefore shown only at times when both a donor and acceptor fluorophore are present on the DNA molecule. The black: Dex, (Aem + Dem) panel shows donor-excited total emission (see Figure 1—figure supplement 3). An objective image-analysis algorithm (Friedman and Gelles, 2015) detects a spot of fluorescence at time points shown in green, red, and black on the time records. Gray, gray-dash, and yellow highlight three 10-s time intervals referenced in (d). A.U., arbitrary units. Concentrations of labeled proteins in the reaction are 0.5 nM ORC5C-549, 15 nM Cdt1–Mcm2-72C-649. Figure 1—figure supplement 2a shows additional records. (d) Histogram plots of EFRET values for 106 single-ORC-mediated, double-hexamer formation events during three 10-s time intervals: immediately after the first (top) or second Mcm2-7 (bottom) arrives or before the second Mcm2-7 arrives (middle). Examples of these intervals are indicated in (c) with gray, yellow, and gray-dash, respectively. The two dashed lines indicate EFRET values of 0 and 1. NDH, number of double-hexamer formation events and nt, number of signal points. Rare EFRET values below −2 and above +2 were excluded (2/428, 13/428, and 1/428 signal points from the top, middle, and bottom histograms, respectively). Figure 1—source data 1 Primary data for histograms in Figure 1d. https://cdn.elifesciences.org/articles/74282/elife-74282-fig1-data1-v3.xlsx Download elife-74282-fig1-data1-v3.xlsx We focused our studies on SM event records that are consistent with Mcm2-7 double-hexamer formation. Mcm2-7 complexes associated with DNA in a one-at-a-time fashion as described previously (Ticau et al., 2015). A fraction of the DNAs (~28% of total DNAs) showed two stepwise increases in Mcm2-72C-649 fluorescence intensity as expected for formation of the Mcm2-7 double hexamer. Of the DNAs with two Mcm2-7 associations, we limited further analysis to a subset showing two Mcm2-7 complexes that are retained on DNA for 20 or more frames of acquisition (>48 s). These long-lived sequential associations occurred on 12% of DNAs and represent successful Mcm2-7 double-hexamer formation (Ticau et al., 2015). The remaining sequential associations (on 16% of DNAs) were short-lived (<48 s), and we considered these to be unsuccessful instances of helicase loading (Ticau et al., 2015). Monitoring ORC and Mcm2-7 DNA association showed that for most double-hexamer formation events, one ORC molecule loaded two Mcm2-7 complexes. Specifically, we observed association of one ORC molecule with the DNA throughout the interval for sequential recruitment of two Mcm2-7 molecules in 81% of double-hexamer formation events. The observation of only one ORC is not due to incomplete ORC labeling as we determined that 88 ± 2% of the ORC5C-549 protein was labeled (see Materials and methods). Thus, if a second ORC was required for recruiting the two helicases, we would expect to see association of two ORCs in 77% (0.882 = 0.77) of double-hexamer formation events. In contrast, we observed two ORC proteins during sequential Mcm2-7 recruitment in only 13% of the double-hexamer formation events (22/166). The remaining 6% of double-hexamer formation events had three associated ORC molecules, likely reflecting one or more nonspecific ORC DNA-binding events. In the small fraction of double-hexamer formation events with two ORC molecules, the arrival of the second ORC was not coordinated with any other event we could observe during helicase loading. We found two types of two-ORC double-hexamer formation events. In 8/166 events, different ORC molecules were present during the two Mcm2-7 associations. The first ORC appeared to release from DNA following the recruitment of the first Mcm2-7, and a second ORC was present during the recruitment of the second Mcm2-7. These rare events provide the first direct evidence of helicase loading mediated by two separate ORC molecules. For 14/166 events, one ORC molecule was present throughout and an additional, second ORC associated between the first and second Mcm2-7 recruitment events. We note that for this category, it is possible that a single ORC molecule mediates both recruitment events with the second ORC being DNA bound but uninvolved (see ‘The same ORC molecule interacts with both Mcm2-7 helicases’). We restricted subsequent analysis to the one ORC double-hexamer formation events for two reasons. First, they were the most frequent events observed. Second, we were particularly interested in investigating how one ORC could load two Mcm2-7 helicases in opposite orientations. FRET between ORC5C-549 and Mcm2-72C-649 is a reporter for C-terminal ORC–Mcm2-7 interactions during helicase recruitment (Figure 1b). When both ORC5C-549 and Mcm2-72C-649 were colocalized with DNA, we observed transient periods of high EFRET upon Mcm2-7 DNA association (Figure 1c, blue: Dex, [Aem/(Aem + Dem)] panel). Mean EFRET ± standard error of the mean (SEM) in the 10-s intervals after first and second Mcm2-7 arrivals were 0.72 ± 0.01 and 0.79 ± 0.01, respectively (Figure 1d, top and bottom panels). Three findings give us confidence that these high EFRET values arise from interactions between ORC and Mcm2-7. First, we positioned the fluorescent probes on ORC and Mcm2-7 to detect interactions between regions that are proximal in the complex presumed to represent the initially recruited Mcm2-7, the OCCM (Yuan et al., 2017). Second, in 105/111 instances where a first Mcm2-72C-650 arrived at an ORC5C-549-bound DNA, high EFRET was detected at the same time as the arrival of the Mcm2-7 on DNA (temporal resolution is ±2.4 s; Figure 1—figure supplement 2b, c). This coordination indicates that the high EFRET value monitors an interaction occurring during initial Mcm2-7 recruitment. Finally, we verified that observing high EFRET was dependent on the placement of the fluorescent probes on Mcm2-7 and ORC. Moving the label on either ORC or Mcm2-7 to the opposite side of the protein relative to the C-terminal ORC–Mcm2-7 interface seen in the OCCM resulted in strong reductions in the associated EFRET values (Figure 2—figure supplement 1, Figure 3—figure supplement 1). Together, these observations establish that the high EFRET state arises from specific ORC–Mcm2-7 interactions during initial recruitment of Mcm2-7. We note that the total emission upon donor excitation is enhanced during OM interactions (Figure 1c, black: Dex, (Dem + Aem) panel). This is due to protein-induced fluorescence enhancement (Hwang et al., 2011) of the of the DY549P1 fluorophore during the OM interaction (Figure 1—figure supplement 3). Importantly, this effect does not hinder our ability to detect OM interactions. Instead, it represents an additional indicator of this interaction. Each Mcm2-7 recruitment is accompanied by an OM interaction Monitoring the ORC–Mcm2-7 recruitment interface revealed two periods of OM interaction during helicase loading. Arrival of each of the two Mcm2-7 helicases was accompanied by a short period of high EFRET in almost all cases (Figure 1c, blue: Dex, [Aem/(Aem+Dem)] panel). In 111 examples of double-hexamer formation, 106 showed distinct periods of high EFRET with each of the two Mcm2-7 arrivals (see below for a description of the remaining 5/111 cases). The elevated EFRET values were simultaneous with the arrival of the corresponding Mcm2-7 on the DNA (Figure 1—figure supplement 2c), consistent with both OM interactions mediating Mcm2-7 recruitment. The start time of the first OM interaction matched the first Mcm2-7 arrival in 104/106 cases and the start of the second OM interaction matched the arrival of the second Mcm2-7 in 98/106 cases (time resolution of experiment ~ 2.4 s). The rare cases (2/106 and 7/106 for the first and second Mcm2-7, respectively) that were not simultaneous were all within 4.8 s of the corresponding Mcm2-7 arrival except for one (6 s). The similar mean EFRET values associated with the first and second OM interactions (0.72 ± 0.01 and 0.79 ± 0.01, respectively; Figure 1d) indicate that recruitment of the first and second Mcm2-7 is mediated by the formation of a similar ORC–Mcm2-7 interface. The first OM interaction consistently ended before the second Mcm2-7 was recruited during helicase loading. Although both ORC and first Mcm2-7 remained associated with the DNA, the OM-FRET signal was lost before second Mcm2-7 arrival in 106/106 cases. Thus, there was always a period lacking OM interaction between the two Mcm2-7 recruitment events. Consistent with this observation, EFRET values in the 10-s interval preceding the second Mcm2-7 arrival were low (Figure 1d, middle panel), with an average value of 0.08 ± 0.02. The second OM interaction observed was also short lived and in a majority of events ended with the loss of ORC5C-549 fluorescence (71/106; Figure 1—figure supplement 4a). We confirmed this loss of donor fluorescence was typically not due to photobleaching (Figure 1—figure supplement 4b), indicating that the end of the second OM interaction in these cases was concomitant with the departure of ORC from the DNA. These data indicate that the interactions that recruit the first Mcm2-7 are always broken before the second Mcm2-7 is recruited, as expected if the same ORC mediated both events. ORC interacts with both the first and second Mcm2-7 during double-hexamer formation Two distinct mechanisms of helicase loading could result in the observation of two sequential OM interactions with a single ORC protein. The two high EFRET signals could arise due to ORC5C-549 interacting twice with the first Mcm2-72C-649 (Figure 2a, Model 1, ‘Re-FRET’). Alternatively, ORC5C-549 may instead interact with each of the two Mcm2-72C-649 complexes on arrival at the DNA (Model 2). To achieve the head-to-head conformation of the Mcm2-7 helicases in the double hexamer, Model 2 would require either ORC or the first Mcm2-7 to invert its orientation on the DNA. Since initial loading of the Mcm2-7 complex involves passing origin DNA through the Mcm2-5 gate followed by gate closing, inversion of Mcm2-7 on the DNA would require reopening of the Mcm2-5 gate. However, previous studies found no evidence of Mcm2-5 gate reopening during helicase loading (Ticau et al., 2017). For this reason, the version of Model 2 in which ORC rebinds the DNA on the opposite side of the first Mcm2-7 and in the opposite orientation is most likely. We call this the ‘ORC-flip’ model. Figure 2 with 2 supplements see all Download asset Open asset ORC makes OM interactions sequentially with the first and second Mcm2-7 helicases. (a) Two models to explain an OM-FRET interaction accompanying each Mcm2-7 arrival in an experiment (Figure 1) in which both ORC and Mcm2-7 are C-terminally labeled. Both models begin with ORC recruiting the first Mcm2-7 via the first OM interaction (high EFRET). In the Re-FRET model, ORC separates from the first Mcm2-7 (resulting in low EFRET) followed by reinteracting with the first Mcm2-7. In the ORC-flip model, ORC releases from its original binding site and rebinds the DNA at a second inverted binding site on the other side of Mcm2-7, resulting in lower EFRET. The flipped ORC then recruits the second Mcm2-7 via a second OM interaction. (b) Experimental setup to distinguish between the Re-FRET and ORC-Flip models. Acceptor-labeled Mcm2-72C-649 (C; red circles) and Mcm2-74N-650 (N; red circles) are mixed in an equimolar ratio, resulting in four subpopulations of double hexamers – CC, NN, CN, and NC. Red dashed lines indicate Mcm2-7 arrival times during double-hexamer formation. Only the C-terminally labeled Mcm2-72C-649 molecules exhibit high FRET with donor-labeled ORC5C-549. The Re-FRET and ORC-flip models predict distinct FRET profiles for the four double-hexamer populations generated. Importantly, the observation of single FRET peaks is unique to the ORC-flip model. (c) Representative double-hexamer formation events from an experiment with mixed Mcm2-72C-649 and Mcm2-74N-650. Concentrations of labeled proteins in the reaction are 0.5 nM ORC5C-549, 7.5 nM Cdt1–Mcm2-72C-649 and 7.5 nM Cdt1–Mcm2-74N-650. Figure 2—figure supplement 2 shows additional records of CN and NC double hexamers. (d) The fraction of observed events (± standard error, SE) corresponding to the type of FRET profile shown above in (c). Figure 2—source data 1 Primary data for graph in Figure 2d. https://cdn.elifesciences.org/articles/74282/elife-74282-fig2-data1-v3.xlsx Download elife-74282-fig2-data1-v3.xlsx To distinguish between the Re-FRET and ORC-flip models, we performed the SM helicase-loading reaction with a mixture of two Mcm2-7 preparations labeled at different positions with acceptor fluorophores (Mcm2-72C-649 and Mcm2-74N-650). Importantly, in contrast to Mcm2-72C-649, the N-terminally labeled Mcm2-74N-650 exhibits very low FRET with ORC5C-549 (Figure 2—figure supplement 1). Mixing an equimolar ratio of these two modified Mcm2-7 complexes with ORC5C-549 allows the formation of four distinct populations of double hexamers (Figure 2b). We will refer to the four populations of double hexamers by the position of the acceptor fluorophore on the first and second helicases in that order. Thus, a double hexamer formed where the first helicase is Mcm2-72C-649 and second is Mcm2-74N-650 will be referred to as ‘CN’ and a double hexamer where the first helicase is Mcm2-74N-650 and second is Mcm2-72C-649 is ‘NC’. Double hexamers with the same labeled Mcm2-7 for the first and second events would be ‘CC’ or ‘NN.’. Depending on which mechanism is used, the four double-hexamer populations will generate distinct OM interaction profiles (Figure 2b). If the Re-FRET model is accurate, ORC would only interact with the first recruited helicase. Since the CC and CN double hexamers have Mcm2-72C-649 as the first helicase, these populations should have two sequential high FRET peaks corresponding to each helicase arrival. In contrast, because the NN and NC double hexamers have Mcm2-74N-650 as the first helicase, the Re-FRET model predicts these populations should not exhibit high FRET during double-hexamer formation. If the ORC-flip model is correct, ORC would form an OM interaction sequentially with each Mcm2-7 helicase in the double hexamer. Thus, for this model only CC double hexamers should be associated with two sequential high FRET peaks. Importantly, for the ORC-flip model the mixed double hexamers (CN and NC) would exhibit a single high FRET peak when the C-terminally labeled Mcm2-72C-649 arrives. Thus, the observation of single OM interaction EFRET (OM-FRET) peaks during loading of two helicases is unique to the ORC-flip model, allowing us to distinguish between the two models. We observed four distinct patterns of OM interaction profiles at similar frequencies when the differently labeled Mcm2-7 complexes were present (Figure 2c, Figure 2—figure supplement 2). Of 90 double-hexamer formation events, we identified 25 events with 2 OM-FRET peaks and 21 events with no associated OM-FRET. Strikingly, approximately half the double-hexamer formation events had a single OM-FRET peak associated with them (44/90), consistent with the ORC-flip model (see Figure 2b, CN and NC panels). Also consistent with this model, these single OM-FRET events are equally distributed between events in which FRET is associated with the first (22/90 events) or the second (22/90 events) Mcm2-7 arrival (Figure 2d). Indeed, based on the EFRET patterns, we were able to infer the underlying double-hexamer ‘type’ for each OM-FRET pattern revealing similar frequencies of CC, NN, CN, and NC double hexamers. Both the presence of the four patterns of FRET and their equal frequency strongly support the ORC-flip model. We note that these patterns cannot be explained by incomplete labeling of Mcm2-7 complexes, as we only analyzed events with two labeled Mcm2-7 and a labeled ORC. In the experiment with only C-terminally labeled Mcm2-7 (Figure 1), most events exhibited two sequential OM-FRET peaks, but rare events did not. We observed 1/111 events in which only the first Mcm2-7 arrival exhibited FRET and 3/111 events in which only the second Mcm2-7 arrival exhibited FRET. We also observed 1/111 events with no FRET on either Mcm2-7 arrival. Although all five events appear to have a single ORC colocalized with DNA, these events are likely to arise from DNAs with two bound ORC molecules, one of which is unlabeled. Importantly, these events occur at very low frequency and cannot explain the frequent observation of one or no FRET peaks observed in reactions with mixed Mcm2-72C-649 and Mcm2-74N-650 complexes (Figure 2d). Thus, we conclude that ORC makes OM interactions with both the first and second Mcm2-7 helicases during helicase loading. The same ORC molecule interacts with both Mcm2