Unwinding of a eukaryotic origin of replication visualized by cryo-EM

Abstract

To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA. Here the authors used cryogenic electron microscopy and biochemistry to understand how yeast Mcm10 exerts its essential role in DNA replication initiation, finding that it splits the double Cdc45-MCM-GINS-Polε structure. The lagging-strand template is ejected from each MCM ring as the central channel of the helicase becomes too tight to accommodate two DNA strands.