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

Sarah S Henrikus; Marta H Gross; Oliver Willhoft; Thomas Pühringer; Jacob S Lewis; Allison W McClure; Julia F Greiwe; Giacomo Palm; Andrea Nans; John F X Diffley; Alessandro Costa

doi:10.1038/s41594-024-01280-z

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

Nat Struct Mol Biol. 2024 May 17. doi: 10.1038/s41594-024-01280-z. Online ahead of print.

Authors

Affiliations

¹ Macromolecular Machines Laboratory, Francis Crick Institute, London, UK.
² Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
³ Chromosome Replication Laboratory, Francis Crick Institute, London, UK.
⁴ Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
⁵ Structural Biology Science Technology Platform, Francis Crick Institute, London, UK.
⁶ Macromolecular Machines Laboratory, Francis Crick Institute, London, UK. alessandro.costa@crick.ac.uk.

PMID: 38760633
DOI: 10.1038/s41594-024-01280-z

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.