A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

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 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. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession 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.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

“…In this model, ACS confers origin specificity in S.c. by facilitating ORC binding to nucleosomes proximal to a nucleosome-free ARS sequence. We note that MCM DHs are recruited to origins via a "single ORC flipping" or "two ORC" mechanism[10][11][12][13] , and that our model is compatible with either scenario.…”

“…ORC works in concert with Cdc6 and Cdt1 to coordinate the loading of two Mcm2-7 helicase complexes (MCM) onto the origin DNA 8,9 , forming the MCM double hexamer (DH), or "pre-replication complex" (pre-RC). Recent reports demonstrate that the two MCM hexamers are loaded onto DNA by two ORCs or by one ORC that flips between two elements within the origin [10][11][12][13] . Only a fraction of MCMs loaded on chromatin become activated during the subsequent S phase to produce bidirectional replication forks.…”