[PDF][PDF] An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly

A Fernández-Cid, A Riera, S Tognetti, MC Herrera… - Molecular cell, 2013 - cell.com
A Fernández-Cid, A Riera, S Tognetti, MC Herrera, S Samel, C Evrin, C Winkler, E Gardenal…
Molecular cell, 2013cell.com
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 …
Summary
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.
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