A key event in eukaryotic DNA replication is origin licensing in G1-phase, during which two Mcm2-7 replicative DNA helicases are loaded onto each origin DNA in an inactive, head-to-head fashion. Origin licensing marks every potential origin in a cell, and the opposing orientation of the loaded helicases ensures that they are poised to initiate bidirectional replication when the cell enters S-phase. Although it has long been known that the origin-recognition complex (ORC) binds origin DNA to direct helicase loading, the molecular mechanism by which two oppositely oriented helicases are loaded remains puzzling. Previous biochemical studies found evidence in support of a two-ORC mechanism for helicase loading wherein each of the two Mcm2-7 helicases are recruited by a separate, oppositely oriented ORC molecule. In contrast, single-molecule and cryo-EM approaches observed predominantly one ORC involved in helicase loading, but could not explain how a single ORC could load two oppositely oriented helicases.

In this paper, a collaboration with Steve Bell’s lab at MIT, Ph.D. student Shalini Gupta reconciles these seemingly contradictory observations. Using single-molecule fluorescence energy transfer (sm-FRET), she observed interactions in vitro between individual ORC molecules and the Mcm2-7 helicases in real time at two separate interfaces. In the large majority of instances, a single ORC molecule recruits both Mcm2-7 helicases through direct interactions. Between recruitment of the first and the second helicase, ORC ‘flips’ its orientation on DNA using a flexible protein tether to the first loaded Mcm2-7. This remarkable ORC inversion ensures that the two helicases are recruited via similar interactions, but in opposite orientations. The data define a complete, integrated pathway for helicase loading that resolves the apparent contradictions between previous observations. The tethered-flip mechanism provides a molecular explanation for how cells avoid the potentially damaging consequences of incompletely-formed helicase pairs at origins.

This article was the subject of an eLife “Insight article” by Bruce Stillman.