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          Institute: MPI für molekulare Genetik     Collection: Microscopy     Display Documents



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ID: 473602.0, MPI für molekulare Genetik / Microscopy
A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.
Authors:Evrin, Cecile; Clarke, Pippa; Zech, Juergen; Lurz, Rudi; Sunc, Jingchuan; Uhle, Stefan; Li, Huilin; Stillman, Bruce; Speck, Christian
Language:English
Date of Publication (YYYY-MM-DD):2009-12-01
Title of Journal:Proceedings of the National Academy of Sciences of the United States of America
Journal Abbrev.:Proc. Natl. Acad. Sci. U S A
Volume:106
Issue / Number:48
Start Page:20240-
End Page:20245
Copyright:2009 by the National Academy of Sciences
Review Status:not specified
Audience:Experts Only
Abstract / Description:During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.
Comment of the Author/Creator:To whom correspondence may be addressed.
E-mail: stillman@cshl.edu or christian.speck@csc.mrc.ac.uk.

This article contains supporting information online at www.pnas.org/cgi/content/full/0911500106/DCSupplemental.
External Publication Status:published
Document Type:Article
Communicated by:Rudi Lurz
Affiliations:MPI für molekulare Genetik
External Affiliations:1.DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom;
2.Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA;
3.dCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Identifiers:URL:This article contains supporting information onlin...
DOI:10.1073/pnas.0911500106
ISSN:0027-8424
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