title: Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2-7 helicases.
abstract: 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.
authors: Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors: Thomas J
authors: TJ
authors: Takara
authors: Stephen P
authors: SP
authors: Bell
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C069200:MCM5 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; D004265:DNA Helicases; C069199:MCM2 protein, S cerevisiae; C507761:MCM3 protein, S cerevisiae; C092802:MCM4 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7