A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.
Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.
Pasero
P
P
Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges/Lausanne, Switzerland.
Duncker
B P
BP
Schwob
E
E
Gasser
S M
SM
D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004271:DNA, Fungal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000502:physiology*; D016193:G1 Phase; D051738:Origin Recognition Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae