Regulation of replication origin licensing by ORC phosphorylation reveals a two- step mechanism for Mcm2-7 ring closing

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin- dependent kinase (CDK) phosphorylation of three helicase- loading proteins: Cdc6, the Mcm2- 7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2- 7 is well understood. Here we use single- molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2- 7 to origins but prevents second Mcm2- 7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2- 7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase- loading protein. Real- time monitoring of first Mcm2- 7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2- 7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed- ring form of Mcm2- 7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2- 7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2- 7 ring is a two- step process started by Cdt1 release and completed by MO complex formation.

Automated summary

Eukaryotic DNA replication occurs only once per cell cycle, which is regulated by the temporal separation of helicase loading and activation. Phosphorylation of ORC by CDK inactivates helicase loading by preventing the recruitment of a second Mcm2-7 helicase and destabilizing the first Mcm2-7 ring closure. This phosphorylation also inhibits the formation of the MO complex, which is necessary for stable closure of the first Mcm2-7. These findings demonstrate that ORC phosphorylation affects multiple steps of helicase loading and elucidate the two-step process of the first Mcm2-7 ring closure.