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A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.

ABSTRACT: 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.

SUBMITTER: Evrin C

PROVIDER: S-EPMC2787165 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.

Evrin Cecile C Clarke Pippa P Zech Juergen J Lurz Rudi R Sun Jingchuan J Uhle Stefan S Li Huilin H Stillman Bruce B Speck Christian C

Proceedings of the National Academy of Sciences of the United States of America 20091112 48

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 stud ...[more]

PMID: 19910535

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Project description:Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.Significance statementEach time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.

Dataset Information

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.

Publications

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.

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