Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ( 5 S and 53 S) and at least three minor phosphorylation sites ( 4 S, 7 S, and 59 T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of 5 S and 53 S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19 -30 and containing 26 S and 27 S), it phosphorylated 26 S efficiently when this peptide contained a chemically synthesized phospho-27 S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclindependent and Cdc7 kinases is essential for the initiation of DNA replication.replication ͉ prereplicative complex ͉ origin activation C dc7 plays an essential role in the initiation of eukaryotic DNA replication (1-3). Cdc7 encodes a serine͞threonine kinase that is highly conserved from yeast to human (4). The activity of this kinase fluctuates during the cell cycle and depends completely on the regulatory subunit, Dbf4. Dbf4 accumulates during the S and G 2 phases of the cell cycle and is degraded rapidly during the G 1 phase by the anaphase promoting complex (5, 6). In Saccharomyces cerevisiae, Dbf4 binds to chromatin at the G 1 ͞S transition and remains on chromatin during the S phase (7). In the Xenopus egg extract cell-free system, Cdc7 was found to bind to chromatin during S phase, and this association required the minichromosome maintenance (MCM) complex (8).Both genetic and biochemical studies in S. cerevisiae indicate that Cdc7 kinase activity is required throughout S phase to activate replication origins on chromosomes (9). Cdc7 kinase acts directly on individual replication origins, presumably by phosphorylating components of the prereplicative complex (pre-RC), which may lead to the remodeling of the pre-RC for the unwinding of replication origins and subsequent recruitment of the replication fork machinery. Although the mechanism-oforigin activation by Cdc7 kinase remains unclear, the MCM complex appears to be the primary physiological target of this kinase activity (2).The MCM complex is composed of six distinct subunits (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, and Mcm7), which are all related structurally and highly conserved in eukaryotes (10). All six proteins are essential for the initiation of DNA replication and contain sequence motifs required for DNA helicase activity. In keeping with this notion, the MCM subcomplex containing Mcm4, Mcm6, and Mcm7 posses...