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Vol. 13, No. 16, pp. 2159-2176, August 15, 1999
1 Swiss Institute for Experimental Cancer Research (ISREC),
CH-1066 Epalinges/Lausanne, Switzerland; 2 Institute
of Molecular Genetics, UMR 5535, Centre National de la Recherche
Scientifique, F-34033 Montpellier, France
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 G1-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 G1 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 G1-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
G1/S transition, by a pathway parallel to, but
independent of, the Cdc6p-dependent recruitment of MCMs.
[Key Words: DNA replication; CDC28; CDC7; DBF4; S-phase-promoting factor]
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