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Genes Dev. 2014 Aug 1;28(15):1653-66. doi: 10.1101/gad.242404.114.

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.

Author information

1
DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
2
Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA;
3
Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA.
4
DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom; chris.speck@imperial.ac.uk.

Abstract

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.

KEYWORDS:

DNA licensing; DNA replication; cancer; genomic stability; pre-RC; replicative helicase

PMID:
25085418
PMCID:
PMC4117941
DOI:
10.1101/gad.242404.114
[Indexed for MEDLINE]
Free PMC Article

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