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TitleUnwinding of a eukaryotic origin of replication visualized by cryo-EM.
Journal, issue, pagesNat Struct Mol Biol, Year 2024
Publish dateMay 17, 2024
AuthorsSarah 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 /
PubMed AbstractTo 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 ...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.
External linksNat Struct Mol Biol / PubMed:38760633
MethodsEM (single particle)
Resolution3.7 - 4.5 Å
Structure data

EMDB-17449, PDB-8p5e:
S. cerevisiae nexus-sCMGE after DNA replication initiation
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-17458, PDB-8p62:
S. cerevisiae ssDNA-sCMGE after DNA replication initiation
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-17459, PDB-8p63:
S. cerevisiae consensus-sCMGE on ssDNA after DNA replication initiation
Method: EM (single particle) / Resolution: 3.7 Å

EMDB-17460: S. cerevisiae sCMGE with N-ter Mcm10 density
Method: EM (single particle) / Resolution: 4.5 Å

Chemicals

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

ChemComp-ZN:
Unknown entry

ChemComp-MG:
Unknown entry

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

Source
  • saccharomyces cerevisiae (brewer's yeast)
  • synthetic construct (others)
  • DNA molecule (others)
KeywordsREPLICATION / Saccharomyces cerevisiae / helicase / CMGE / initiation of DNA replication / DNA / DNA unwinding

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