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. 2025 Oct 14;122(41):e2510685122.
doi: 10.1073/pnas.2510685122. Epub 2025 Oct 7.

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

David Driscoll  1   2 , Larry J Friedman  2   3 , Jeff Gelles  3 , Stephen P Bell  1   2
Affiliations

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

David Driscoll et al. Proc Natl Acad Sci U S A. .

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 region of Mcm2 during ORC's 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.

Keywords: DNA replication initiation; Mcm2-7 helicase; origin licensing; origin recognition complex (ORC); single-molecule FRET.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Orc6N interacts specifically with Mcm2-7N rapidly upon Mcm2-7 recruitment. (A) Schematic of the single-molecule colocalization/FRET assay used in this study. Blue-excited fluorophore-labeled DNA molecules containing an origin of replication were attached to a microscope slide. A solution containing all four helicase-loading proteins (labeled as indicated, stars) was incubated with DNA. Colocalization of green (donor) or red (acceptor) fluorophores with blue fluorophores was used as a proxy for protein–DNA associations (32). (B) Detection of Orc6N:Mcm2-7N interaction by single-molecule FRET. In the experiment shown (A), ORC is labeled with a donor fluorophore (green star) adjacent to its N-terminal domain (ORC6N-550) and Mcm2-7 is labeled with an acceptor fluorophore (red star) at the N terminus of Mcm6 (Mcm2-76N-650). When both labeled proteins are present on a DNA (A, Right), a low EFRET is expected when Orc6N and Mcm2-7N do not directly interact (Left) and elevated EFRET is expected when Orc6N and Mcm2-7N interact (Right). (C) Representative traces showing ORC6N-550 and Mcm2-76N-650 DNA associations and Orc6N:Mcm2-7N interactions. Top plot: acceptor emission during acceptor excitation (red, Aex, Aem). The red dashed line indicates time of Mcm2-76N-650 recruitment to DNA. Second plot: Donor emission during donor excitation (green, Dex, Dem). The green dashed line indicates time of ORC6N-550 binding to DNA. Third plot: FRET, acceptor emission during donor excitation (pink, Dex, Aem). Fourth plot: total emission during donor excitation [black, Dex, (Dem + Aem)]. Bottom plot, effective FRET efficiency (EFRET)(blue, Dex, Aem/(Dem + Aem)). The blue dashed line indicates the transition from a low EFRET state to a high EFRET state corresponding to Orc6N:Mcm2-7N interactions. Gray line segments represent background signal when no fluorescent protein is colocalized with the DNA molecule. Line segments with signal above background are colored and indicate bound protein. Inset highlights EFRET values (blue) after Mcm2-7 arrival (red dashed line). The horizontal black dashed line inside inset represents the Orc6N:Mcm2-7N EFRET threshold value (see SI Appendix, Fig. S2 and SI Methods for details). The blue dashed vertical line in Inset represents time at which EFRET values move from below to above threshold value. (D) EFRET distribution heat map for 201 DNA molecules bound by ORC6N-550 and Mcm2-76N-650. Time is after Mcm2-76N-650 arrival. The EFRET probability density (color scale) was calculated using only the DNA molecules with bound ORC6N-550 and Mcm2-76N-650 that remained at each time point; the fraction of remaining complexes (blue curve) and 95% CI (orange shading) are shown in the bottom plot. The dashed line is the threshold value used to distinguish between low and high EFRET states (see SI Appendix, Fig. S2 and SI Methods for details).
Fig. 2.
Fig. 2.
Mcm2N, Orc6N, and the adjacent Orc6 linker region facilitate the Orc6 tether interaction. (A) Schematic diagram of the Mcm2 and Orc6 mutants used in these experiments. The black oval or line represents the scrambled mutant of Mcm2 or the lnk1 region of Orc6, respectively. See SI Appendix, Fig. S4A for mutant and labeling details. (B) Heat maps showing the probability density of Orc6 tether interactions over time after Mcm2-7 arrival using the mutant constructs: Mcm2-72scr-6N-650 (Left), ORC6ΔN-550 (Middle Left), ORC6-lnk1scr-550 (Middle Right), and ORC6∆N-lnk1scr-550 (Right). For mutants that allowed tether formation, the dashed line is the threshold value used to distinguish between low and high EFRET states for each construct (SI Appendix, Fig. S2B). Bottom plots show fraction of events that retain both ORC and Mcm2-7 molecules at the indicated times after first Mcm2-7 recruitment (orange shading = 95% CI). (C) Time to first formation of Orc6 tether interaction. Wild type (ORC6N-550, black), ORC6ΔN-550 (red), and ORC6-lnk1scr-550 (blue) are plotted relative to Mcm2-76N-650 arrival. The Y-axis shows the fraction of molecules that formed Orc6 tether interaction (shading = 95% CI). To facilitate temporal comparison, molecules that did not reach a high EFRET state were not included in the analysis. Inset: Bar graph showing median times (±SE) to reach high EFRET state. Mutants that show little or no Orc6-tether interaction (Mcm2-72scr-6N-650 and ORC6∆N-lnk1scr-550) are not included. (D) Cumulative survival curve of initial Orc6 tether interactions. Wild type (ORC6N-550, black), ORC6ΔN-550 (red), and ORC6-lnk1scr-550 (blue) are plotted (shading = 95% CI). Only the first Orc6 tether interaction for a DNA molecule was considered for this analysis. The ORC6ΔN-lnk1scr-550 data were not plotted on the line graph for clarity due to few data points (n = 5). Molecules that never formed the tether interaction were eliminated from the analysis. Inset: Bar graph showing median durations (±SE) of high EFRET state. (E) Percentage of molecules that formed the Orc6 tether interaction. The percentage (±SE) was determined by counting the number of ORC–Mcm2-7 interactions that reached the high EFRET state (defined as two consecutive measurements above threshold) relative to total Mcm2-7 recruitment events.
Fig. 3.
Fig. 3.
Orc6 interacts with Mcm2-7N before Cdt1 release and MO formation, tethering ORC to Mcm2-7 during its binding-site transition. (A) Model for Orc6 tethering during OM to MO transition. Upon Cdt1 departure and breaking of OM interactions, Orc6 retains ORC at site of helicase loading to enable ORC flipping and formation of the MO complex. (B) Experiment design for simultaneous assessment of Orc6 tether interaction and Cdt1 release. ORC6N-550 (donor) and Mcm2-76N-650 (acceptor) are labeled as described previously (Fig. 1). Cdt1C-650 is labeled with an additional acceptor fluorophore (red star). Diagram illustrates the expected sequence of species (Top) and the single-DNA recording that would result if the tether interaction occurs before Cdt1 release (Bottom). Binding of Mcm2-76N-650-Cdt1C-650 causes an increase in red fluorescence. Orc6N and Mcm2-7N interaction results in a low to high EFRET transition (blue plot). Cdt1 release causes a 50% decrease in red fluorescence (top plot) with no change in EFRET. The difference in the time of the latter two events (double-headed arrow) determines when Orc6N and Mcm2-7N interact relative to Cdt1 release. (C) Representative single DNA molecule record from the experiment outlined in panel B. Traces are arranged as follows: Top plot: acceptor emission during acceptor excitation (red, Aex, Aem). The first red dashed line indicates time of first Mcm2-76N-650-Cdt1C-650 recruitment to DNA, and the second red dashed line indicates release of first Cdt1C-650. The third red dashed line indicates second Mcm2-76N-650-Cdt1C-650 recruitment, and the fourth red dashed line indicates second Cdt1C-650 release. Second plot: Donor emission during donor excitation (green, Dex, Dem). The first green dashed line indicates time of ORC6N-550 binding to DNA. The second green dashed line indicates time of ORC6N-550 release. Bottom plot, effective FRET efficiency (EFRET)(blue, Dex, Aem/(Dem + Aem)). The blue dashed line indicates the transition from a low EFRET state to a high EFRET state corresponding to Orc6 tether interactions. See SI Appendix, Fig. S7C for additional records. (D) Histogram of times between Cdt1 release and the time of first Orc6N interaction with Mcm2-7N. Of 162 events that recruited both ORC6N-550 and Mcm2-76N-650-Cdt1C-650, 152 events where a high EFRET interaction was observed are shown. (E) Survival plot showing fraction of ORC molecules retained after Cdt1 release. Data were segregated by whether ORC6N-550 and Mcm2-76N-650 had a high (black) or low (red) EFRET value at the time of Cdt1 release. DNA molecules where ORC was not detected at the time of Cdt1 release (15/162) were excluded from this analysis. Inset shows 0 to 50 s after Cdt1 release to highlight differences in early time points. (F) Impact of Orc6 mutants on ORC retention after Cdt1 release. Survival plots are shown for WT ORC and ORC containing Orc6 mutations (black: WT ORC6N-550, red: ORC6∆N-550, blue: ORC6-lnk1scr-550, purple: ORC6ΔN-lnk1scr-550). In contrast to E, the data are not separated by EFRET state at time of Cdt1 release. DNA molecules where Mcm2-7 released before ORC (5/111 for ORC6∆N-550, 10/116 for ORC6ΔN-lnk1scr-550) were excluded from this analysis.
Fig. 4.
Fig. 4.
Phosphorylation of ORC inhibits the Orc6 tether interaction. (A) Two representative single DNA molecule records of Orc6-tether assay using phosphorylated ORC6N-550, plotted as described in Fig. 3C. (B) Heat map showing the probability density of Orc6 tether interactions after Mcm2-76N-650 arrival using ORC6N-550 phosphorylated by CDK. Lower plot shows the fraction of molecules remaining and 95% CI (blue curve, orange shading). (C) Time to first formation of the Orc6 tether interaction. Unphosphorylated (black, same data as SI Appendix, Fig. S1E) and phosphorylated (red) ORC6N-550 are plotted relative to Mcm2-76N-650 arrival. ORC was phosphorylated by CDK as described (11). Shading represents 95% CI. To facilitate temporal comparison, molecules that never formed the tether interaction were eliminated from the analysis. Median time to first formation (±SE) of interaction for ORC6N-550 -CDK (black) and ORC6N-550 +CDK (red) (Inset) was determined as in Fig. 2C. (D) Cumulative survival curve of initial Orc6 tether interactions for unmodified (ORC6N-550 –CDK, black) or CDK-modified ORC (ORC6N-550 +CDK, red). Shading represents 95% CI. Molecules that never formed the Orc6 tether interaction were eliminated from the analysis. Only the first interaction for a DNA molecule was considered for this analysis. Median durations of interaction (±SE) were determined as in Fig. 2D. (E) Percentage of molecules that formed the tether interaction are plotted. This percentage (±SE) was determined by counting the number of Orc6 tether interactions that reached the high EFRET state compared to total Mcm2-7 recruitment events.
Fig. 5.
Fig. 5.
Orc6 lnk1 is important for stable second Mcm2-7 recruitment. (A) Single-molecule FRET assay for MO complex formation. Reactions were performed as described in ref. using either WT (ORC6C-550) or ORC containing the Orc6 lnk1scr mutation (ORC6C-lnk1scr-550). ORC was labeled at Orc6 C-terminus (ORC6C-550) and Mcm2-7 was labeled at Mcm3 N terminus (Mcm2-73N-650, 25). (B) Mutations in the Orc6 lnk1 region result in reduced MO complex formation. Bar graph showing the percentage (±SE) of 1st Mcm2-73N-650 binding events that resulted in formation of the MO complex for WT ORC6C-550 (black) and ORC6C-lnk1scr-550 (blue). (C) Representative traces of MO formation assay experiment using ORC6C-550. Plots are arranged as described in Fig. 3C. (D) Representative traces of MO formation assay experiment using ORC6C-lnk1scr-550. Plots are arranged as in C. Red arrowheads indicate failed Mcm2-7 recruitment attempts. (E) Mutations in the Orc6 lnk1 region cause severe defects in double-hexamer formation. Bar graph showing the percentage (±SE) of 1st Mcm2-73N-650 binding events that resulted in formation of an Mcm2-7 double hexamer for WT ORC6C-550 (black) and ORC6C-lnk1scr-550 (blue).
Fig. 6.
Fig. 6.
Model for Orc6 tether function during helicase loading. We propose that Orc6N interacts with Mcm2-7N rapidly after Mcm2-7 recruitment. This enables ORC to remain bound to the site of helicase loading when Cdt1 release occurs and OM interactions are broken, and tethers ORC to Mcm2-7 during its flip over Mcm2-7 to form the MO complex. See text for additional discussion.
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