Abstract
High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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DNA Replication*
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DNA, Fungal / chemistry
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DNA, Fungal / genetics
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DNA, Fungal / metabolism
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Minichromosome Maintenance Complex Component 3 / chemistry
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Minichromosome Maintenance Complex Component 3 / genetics
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Minichromosome Maintenance Complex Component 3 / metabolism
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Minichromosome Maintenance Complex Component 4 / chemistry
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Minichromosome Maintenance Complex Component 4 / genetics
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Minichromosome Maintenance Complex Component 4 / metabolism
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Minichromosome Maintenance Complex Component 6 / chemistry
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Minichromosome Maintenance Complex Component 6 / genetics
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Minichromosome Maintenance Complex Component 6 / metabolism
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Minichromosome Maintenance Complex Component 7 / chemistry
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Minichromosome Maintenance Complex Component 7 / genetics
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Minichromosome Maintenance Complex Component 7 / metabolism
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
Substances
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Cell Cycle Proteins
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DNA, Fungal
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MCM5 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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MCM2 protein, S cerevisiae
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MCM3 protein, S cerevisiae
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MCM4 protein, S cerevisiae
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MCM6 protein, S cerevisiae
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MCM7 protein, S cerevisiae
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Minichromosome Maintenance Complex Component 3
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Minichromosome Maintenance Complex Component 4
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Minichromosome Maintenance Complex Component 6
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Minichromosome Maintenance Complex Component 7