title: Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication.
abstract: Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.
authors.affiliation: Chromosome Replication Laboratory, Cancer Research UK London Research Institute, South Mimms, Herts, UK.
authors.forename: Kin Fan
authors.initials: KF
authors.lastname: On
authors.forename: Fabienne
authors.initials: F
authors.lastname: Beuron
authors.forename: David
authors.initials: D
authors.lastname: Frith
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.forename: Edward P
authors.initials: EP
authors.lastname: Morris
authors.forename: John F X
authors.initials: JF
authors.lastname: Diffley
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology*; D013058:Mass Spectrometry; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018741:Replication Origin / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D004718:Saccharomycetales
chemical_list: D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D064110:Minichromosome Maintenance Proteins