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Multiple Cdt1 molecules act at each origin to load replication‐competent Mcm2–7 helicases
Author(s) -
Takara Thomas J,
Bell Stephen P
Publication year - 2011
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/emboj.2011.394
Subject(s) - biology , replication (statistics) , helicase , genetics , microbiology and biotechnology , virology , computational biology , gene , rna
Eukaryotic origins of replication are selected by loading a head‐to‐head double hexamer of the Mcm2–7 replicative helicase around origin DNA. Cdt1 plays an essential but transient role during this event; however, its mechanism of action is unknown. Through analysis of Cdt1 mutations, we demonstrate that Cdt1 performs multiple functions during helicase loading. The C‐terminus of Cdt1 binds Mcm2–7, and this interaction is required for efficient origin recruitment of both proteins. We show that origin recognition complex (ORC) and Cdc6 recruit multiple Cdt1 molecules to the origin during helicase loading, and disruption of this multi‐Cdt1 intermediate prevents helicase loading. Although dispensable for loading Mcm2–7 double hexamers that are topologically linked to DNA, the essential N‐terminal domain of Cdt1 is required to load Mcm2–7 complexes that are competent for association with the Cdc45 and GINS helicase‐activating proteins and replication initiation. Our data support a model in which origin‐bound ORC and Cdc6 recruit two Cdt1 molecules to initiate double‐hexamer formation prior to helicase loading and demonstrate that Cdt1 influences the replication competence of loaded Mcm2–7 helicases.