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.
Highlights
The eukaryotic DNA replication machinery first assembles and initiates synthesis at DNA sites called origins of replication
To investigate the dynamics of origin-recognition complex (ORC)-Mcm2-7 interactions during helicase loading, we developed a Single-molecule FRET (sm-FRET) assay for the initial interaction between these proteins based on previously described single-molecule helicase-loading experiments (Ticau et al, 2015)
To monitor ORC-Mcm2-7 interactions during loading, purified 179 ORC5C-549, Mcm2-72C-649, Cdt1, and Cdc6 were incubated with fluorescently-labeled surfacetethered origin-DNA
Summary
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 Mcm 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-. The Mcm 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). Mcm in complex with Cdt associates with ORC/Cdc and adjacent DNA to form the short-lived ORC-
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