The structure of ORC\u2013Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6

Xiang Feng,Bruce Stillman,Yasunori Noguchi,Christian Speck,Huilin Li,Marta Barbon

doi:10.1038/s41467-021-24199-1

Abstract

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

Highlights

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication
In the budding yeast Saccharomyces cerevisiae, there are over 400 origins of DNA replication located on 16 chromosomes and they can function as autonomously replicating sequences (ARSs) when inserted into a plasmid[19]
Three components were mixed at a molar ratio of 1 ORC:1.25 DNA:6.4 Cdc[6] in the presence of 5 mM ATPγS under reaction conditions similar to the previously established[14,75] ones

Summary

Results and discussion

In vitro reconstitution and cryo-EM of the ORC–Cdc[6] complex on ARS1 origin DNA. To reconstitute the ORC–Cdc[6] complex, we used an 85 bp fragment of the ARS1 origin DNA containing the A, B1, and B2 elements. Three components were mixed at a molar ratio of 1 ORC:1.25 DNA:6.4 Cdc[6] in the presence of 5 mM ATPγS under reaction conditions similar to the previously established[14,75] ones. Because Cdc[6] binding is known to extend the ORC nuclease protection footprint[14], we used a long DNA in a

TFIIB-A

C35 Arg-267 Orc6-BP

Methods