Unwinding of a eukaryotic origin of replication visualized by cryo-EM
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Sarah Henrikus Marta Gross Oliver Willhoft Thomas Puehringer Jacob Lewis Allison McClure Julia Greiwe Giacomo Palm Andrea Nans John Diffley Alessandro CostaAbstract
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.
Journal details
Volume
31
Issue number
8
Pages
1265-1276
Available online
Publication date
Full text links
Publisher website (DOI)
10.1038/s41594-024-01280-z
Europe PubMed Central
38760633
Pubmed
38760633
