title: Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation.
abstract: The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. Here we address the mechanisms that control the specificity of DDK action. We show that incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, we find that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity.
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Laura I
authors.initials: LI
authors.lastname: Francis
authors.forename: John C W
authors.initials: JC
authors.lastname: Randell
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Takara
authors.forename: Lilen
authors.initials: L
authors.lastname: Uchima
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bell
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C069199:MCM2 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7