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.

Replication of chromosomal DNA in eukaryotes has two major stages.  Starting in the G1 phase of the cell cycle, double hexamers consisting of two copies of the Mcm2-7 replicative helicase are assembled at replication origins.   Later, in S phase, the two helicases are incorporated into two oppositely oriented CMG (Cdc45-Mcm2-7-GINS) complexes that each then form the core of a replisome.  Control of this “activation” step, which is triggered by the protein kinases DDK and S-CDK, is essential to ensure that each part of the genome is replicated once and only once in each cell cycle.

In this paper, Lorraine De Jesús-Kim together with collaborators from Steve Bell’s and Jeff Gelles’ labs used multi-wavelength single-molecule fluorescence colocation  (“CoSMoS”) methods to study in vitro the molecular mechanism of the activation process.  The journal’s acceptance summary notes that “The manuscript provides new and convincing evidence that a heretofore unknown intermediate state [called “CtG”] for replication start contains multiple copies of the GINS and Cdc45 proteins prior to initiation at each origin with one double hexamer of the MCM2-7 complex. The number of GINS and Cdc45 is determined by DDK phosphorylation of the MCM’s and the probability to create an active helicase (CMG) is increased with multiple numbers of the bound ancillary factors…. The single molecule studies and biochemistry are beautifully executed providing the evidence for such intermediates…. The addition of in vivo studies demonstrates that modulating the multiplicity of DDK phosphorylation (and proposed, CtG formation) has an impact on origin usage in cells.”