Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
Date Issued
2014-10-15
Date Acceptance
2014-09-09
Citation
Genes and Development, 2014, 28 (20), pp.2291-2303
ISSN
0890-9369
Publisher
Cold Spring Harbor Laboratory Press
Start Page
2291
End Page
2303
Journal / Book Title
Genes and Development
Volume
28
Issue
20
Copyright Statement
© 2014 Sun et al. This article is distributed exclusively by Cold Spring
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After six months, it is available under a Creative Commons License
(Attribution-NonCommercial 4.0 International), as described at http://
creativecommons.org/licenses/by-nc/4.0/.
Harbor Laboratory Press for the first six months after the full-issue
publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml).
After six months, it is available under a Creative Commons License
(Attribution-NonCommercial 4.0 International), as described at http://
creativecommons.org/licenses/by-nc/4.0/.
License URL
Identifier
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Subjects
Science & Technology
Life Sciences & Biomedicine
Cell Biology
Developmental Biology
Genetics & Heredity
origin recognition complex
DNA replication initiation
replicative helicase
prereplication complex
electron microscopy
EUKARYOTIC DNA-REPLICATION
ORIGIN DNA
ATPASE ACTIVITY
S-PHASE
ORC/CDC6/MCM2-7 COMPLEX
ACTIVE-SITES
HELICASE
INITIATION
HYDROLYSIS
PROTEINS
Publication Status
Published
Date Publish Online
2014-09-09