To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a pre-Replicative Complex (pre-RC) with a MCM2-7 double-hexamer encircling DNA. Using purified components in the presence of ATPγS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the hetero-heptameric ORC-Cdc6 and the hetero-heptameric Cdt1-MCM2-7, called the OCCM complex. Cryo-EM studies of the 14-protein complex reveal that the two separate heptameric complexes are extensively engaged, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. ORC-Cdc6 undergoes a concerted conformational change into a right-handed spiral with the helical symmetry identical to the DNA double helix. The results show a striking structural similarity between the ORC-Cdc6 helicase loader and the Replication Factor-C clamp loader and suggest a conserved mechanism of action.

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.