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 vit ro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is re quired in trans to stimulate initiation in G(1)-phase nuclei, whereas the D bf4/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 detergen t-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cd c6p or Clb/Cdc28 kinase activity, and correlates with competence for initia tion. 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 inde pendent of, the Cdc6p-dependent recruitment of MCMs.