Great insight into the molecular details of cell cycle regulation has been obtained in the past decade. However, most of the progress has been in defining the regulation of the family of cyclin-dependent kinases (CDKs). Recent studies of a myriad of eukaryotic organisms have defined both the regulation and substrates of Cdc7p kinase, which forms a CDK–cyclin-like complex with Dbf4p, is necessary for the initiation of DNA replication and has been conserved in evolution. This kinase is also required for the induction of mutations after DNA damage and for commitment to recombination in the meiotic cell cycle. However, less is known about the role of the kinase in these processes. In a manner similar to CDKs, Cdc7p is activated by a regulatory subunit, Dbf4, the levels of which fluctuate during the cell cycle. One or more subunits of the conserved MCM helicase complex at chromosomal origins of DNA replication are substrates for the kinase during S phase. Phosphorylation of the MCM complex by Cdc7p-Dbf4p might activate DNA replication by unwinding DNA. Therefore, activation of Cdc7p is required for DNA replication. Given that Cdc7p-Dbf4 kinase is overexpressed in many neoplastic cells and tumors, it might be an important early biomarker during cancer progression.

During reproduction, cellular components and organelles need to be replicated and maintained. However, chromosome number needs to be exactly duplicated. Any heritable changes in chromosome number can result in disastrous consequences for the organism. Therefore, elaborate mechanisms to replicate and segregate chromosomes during the cell division cycle have evolved. It has become apparent in the past decade that these mechanisms include reversible protein phosphorylation catalyzed by several protein kinases and phosphatases (see review by Nurse, 2000). Most famous of all these kinases are the cyclin-dependent kinases (CDKs) – see reviews by Morgan, 1995; Roberts, 1999; Sherr and Roberts, 1999). CDKs regulate both the G1-S phase and the G2-M transitions of the cell cycle and probably phosphorylate several different substrates to this end. However, the lesser-known Cdc7p kinase, which like CDKs requires a regulatory subunit (Dbf4p) for activity, has very few protein substrates. Cdc7p-Dbf4p regulates a specific step during the initiation of DNA replication in all eukaryotes studied, including human cells (see recent reviews by Johnston et al., 1999; Masai et al., 1999). Barinaga (1994) reported the emergence of the Cdc7p-Dbf4 complex as an important player in the cell cycle. Here I summarize recent exciting developments that show that this prediction was warranted and elevate the importance of Cdc7p to a level comparable to that of CDKs.

DNA replication

The pioneering work of Hartwell, showed that conditional ts (temperature-sensitive) mutants in cdc7 arrest at the G1-S phase boundary and cannot initiate chromosomal DNA replication at the restrictive temperature, but can complete ongoing DNA replication begun at the permissive temperature (Hartwell, 1973; Hereford and Hartwell, 1974). Furthermore, cdc7 was the only mutant isolated in which the G1-S phase transition was defective that could initiate DNA replication in the absence of protein synthesis. This ‘order of function’ analysis placed the role of Cdc7p close to the initiation of DNA replication. (Hereford and Hartwell, 1974; Fig. 1). One interpretation of this result is that all replication proteins are already made by the time Cdc7p is required and that Cdc7p activates them. However, these early results could not determine whether Cdc7p was needed only to enter S phase or whether Cdc7p was required directly for the initiation of DNA replication. Recently, two groups have provided evidence that Cdc7p acts directly in initiation. They showed that loss of Cdc7p function affects initiation at all origins even that at late origins (Bousset and Diffley, 1998; Donaldson et al., 1998). Therefore, Cdc7p is needed to initiate DNA replication at origins even in cells already in S phase. This role of Cdc7p kinase has been conserved in evolution (Table 1): gene-disruption experiments in fission yeast (Brown and Kelly, 1998; Masai et al., 1995) and antibody inhibition of Cdc7p in frog mitotic extracts (Roberts et al., 1999) and human cells in culture (Jiang et al., 1999; Kumagai et al., 1999) reveal that Cdc7p is also required for DNA replication in other eukaryotes.

Table 1.

Cdc7 and Dbf4 homologues

Cdc7 and Dbf4 homologues
Cdc7 and Dbf4 homologues
Fig. 1.

The order of molecular events in the G1-S phase transition of the cell cycle. (a) The order of the genetic events, as deduced from the original work of Hereford and Hartwell (1974) and based on reciprocal shift analysis of cdc mutants. The step at which protein synthesis is required is indicated. (b) Functions of CDKs and Cdc7p. CDK-Cln phosphorylates Sic1p, which is then targeted for ubiquitin-dependent degradation by Cdc4p. Sic1 is an inhibitor of CDK-Clb complexes. Both Clb proteins and Dbf4p are targeted for degradation by the APC (anaphase-promoting complex).

Fig. 1.

The order of molecular events in the G1-S phase transition of the cell cycle. (a) The order of the genetic events, as deduced from the original work of Hereford and Hartwell (1974) and based on reciprocal shift analysis of cdc mutants. The step at which protein synthesis is required is indicated. (b) Functions of CDKs and Cdc7p. CDK-Cln phosphorylates Sic1p, which is then targeted for ubiquitin-dependent degradation by Cdc4p. Sic1 is an inhibitor of CDK-Clb complexes. Both Clb proteins and Dbf4p are targeted for degradation by the APC (anaphase-promoting complex).

DNA repair

When cells are treated with DNA-damaging agents, they can repair the lesions in a error-free or an error-prone manner. The latter process will produce mutations and is called induced mutagenesis. Cdc7p has a role in induced mutagenesis, as first seen by Njagi and Kilbey (1982). They showed that cdc7ts mutants are sensitive to UV light and mutagenic compounds such as EMS (ethylmethanesulfonate) and nitrogen mustard but exhibit a reduced frequency of both forward and reverse mutations in the presence of these agents. In contrast, levels of spontaneous mutation are unaffected in cdc7 mutants. dbf4 mutants produce a similar result (Ostroff and Sclafani, 1995).

The effects of cdc7 on induced mutation are allele-specific, some alleles displaying higher levels of mutagenesis (Hollingsworth et al., 1992). A complete absence of Cdc7p or Dbf4p is normally lethal, but bob1, a bypass allele of the mcm5 gene (see below), alleviates the need for Cdc7p and hence allows analysis of cdc7Δ mutants (Hardy et al., 1997; Jackson et al., 1993). The bob1 cdc7Δ mutant displays an absence of mutagenesis induced by UV light or EMS, and it is more sensitive to these agents than are the cdc7ts alleles (Pahl, 1994). The cdc7 DNA-repair defect is in the rad6 epistasis group (Kilbey, 1986), which includes genes such as rev1 and rev3 that encode subunits of the error-producing DNA polymerase ζ. Thus, Cdc7p-Dbf4p is important for DNA repair and is essential for induced mutagenesis. However, it is not clear what role Cdc7p plays in these processes. One could speculate that Cdc7p phosphorylates proteins encoded by genes in the rad6 epistasis group and, in some way, activate the error-producing DNA polymerase ζ.

Overproduction of Cdc7p-Dbf4p increases induced mutagenesis by producing more mutations at a given a dose of mutagen (Sclafani et al., 1988). Most induced mutations are produced primarily in cells in S phase (Ostroff and Sclafani, 1995), when Cdc7p-Dbf4p activity is switched on (see below). Therefore, I speculated (Sclafani and Jackson, 1994) that increased levels of Cdc7p-Dbf4p in cancer cells might increase the probability of further oncogenic mutations and thus malignancies. This might account for the improbably high level of mutations needed to produce a malignant cell (Jackson and Loeb, 1998). In this scenario, production of a mutator phenotype by elevated Cdc7p-Dbf4p during tumor progression will increase the chance of producing more mutations. The fact that overexpression of human CDC7 occurs in human cancer cell lines (Hess et al., 1998) and in breast and lung cancer tumor samples from patients (R. A. Sclafani, unpublished; R. Hollingsworth, personal communication) is consistent with this hypothesis. Similar results have been found for ASK, the human homologue of Dbf4p, in cell lines (Kumagai et al., 1999) and in human tumors (R. Hollingsworth, personal communication)

Meiosis

In the meiotic cell cycle, the role of Cdc7p-Dbf4p is less defined. There is clearly a requirement for Cdc7p-Dbf4p after pre-meiotic DNA replication but before DNA recombination (Hollingsworth and Sclafani, 1993; Schild and Byers, 1978). Cdc7-Dbf4 kinase activity is not needed for replication of a plasmid that has a single origin (ARS1) that is used in both mitotic and pre-meiotic DNA replication (Hollingsworth and Sclafani, 1993). This is surprising because Cdc7p-Dbf4p is needed for ARS1 replication in the mitotic cell cycle (Brewer and Fangman, 1987). Homozygous diploid bob1 cdc7Δ mutants can replicate their DNA during meiosis but cannot complete meiosis (Pahl, 1994; Galbraith and R. A. Sclafani, unpublished). In view of recent results of studies of CDKs in meiotic replication, one must interpret these results with caution. An analysis of cdk1ts mutants had shown that Cdk1 is not needed for pre-meiotic DNA replication (Shuster and Byers, 1989), but deletion of the gene that encodes the S phase cyclin Clb5 blocks pre-meiotic DNA replication (Dirick et al., 1998; Stuart and Wittenberg, 1998). Therefore, the cdk1ts mutant might have enough activity under restrictive conditions to allow for DNA replication. Similarly, it is possible that very low levels of Cdc7p-Dbf4p are needed for meiotic DNA replication, but that higher levels are needed after DNA replication. Consistent with this view is the observation that the equivalent of cdc7ts mutants in S. pombe (hsk1ts) do not replicate their DNA during meiosis (S. Forsburg, personal communication). Nonetheless, we still know very little about the role of Cdc7p-Dbf4p after DNA replication in meiosis.

As in the cases of most protein kinases, ‘upstream’ events (kinase regulation) and ‘downstream’ events (kinase substrates) involving Cdc7p can be studied. Amazing progress made on both of these fronts has now given us a clear picture of both events.

Kinase regulation

An analysis of protein sequences has revealed that Cdc7p probably evolved from casein kinase II and has diverged considerably from CDKs (Hunter and Plowman, 1997; Table 1). Our earlier studies had suggested that, although Cdc7p is not a CDK, it can be regulated like a CDK (Fig. 2): the active form contains a stable kinase subunit and an unstable regulatory subunit or cyclin (Jackson et al., 1993; Sclafani and Jackson, 1994). In other words, Dbf4p is the ‘cyclin’ for Cdc7p. Earlier work had shown that ts mutants in DBF4 and CDC7 produce similar phenotypes (Chapman and Johnston, 1989; Hartwell, 1973). Therefore, we hypothesized that the regulatory subunit is Dbf4p (Jackson et al., 1993; Sclafani and Jackson, 1994). We based this idea on the fact that the two proteins interact genetically (Jackson et al., 1993; Kitada et al., 1992) and that immunoprecipitated Cdc7p-Dbf4p is inactive if Dbf4p is inactivated (Jackson et al., 1993). Recently, elegant biochemical studies of both purified endogenous and recombinant proteins support a model for activation of Cdc7 kinase activity by Dbf4p (Brown and Kelly, 1999; Jiang et al., 1999; Lei et al., 1997; Weinreich and Stillman, 1999). Cdc7p-Dbf4p might be a multimeric enzyme that possesses at least two Cdc7p and two Dbf4p subunits (Shellman et al., 1998); S. Forsburg, personal communication).

Fig. 2.

Model for initiation of eukaryotic DNA replication. Both Cdc7p-Dbf4p and Cdk1-Clb are activated in G1 phase and phosphorylate members of the pre-replication complex bound to the ORC at origins. First, Cdc6p is needed to load on the MCM complex. Secondly, Cdk1-Clb is needed for the loading of Cdc45p, which loads on DNA polymerases. Finally, Cdc7p-Dbf4p activates the MCM complex helicase by phosphorylation.

Fig. 2.

Model for initiation of eukaryotic DNA replication. Both Cdc7p-Dbf4p and Cdk1-Clb are activated in G1 phase and phosphorylate members of the pre-replication complex bound to the ORC at origins. First, Cdc6p is needed to load on the MCM complex. Secondly, Cdk1-Clb is needed for the loading of Cdc45p, which loads on DNA polymerases. Finally, Cdc7p-Dbf4p activates the MCM complex helicase by phosphorylation.

The ‘Dbf4 as Cdc7’s cyclin’ model has now been confirmed in many different eukaryotes, including budding yeast (Cheng et al., 1999; Ferreira et al., 2000; Oshiro et al., 1999; Pasero et al., 1999; Weinreich and Stillman, 1999), fission yeast (Brown and Kelly, 1999; Takeda et al., 1999), mouse (Lepke et al., 1999) and humans (Jiang et al., 1999; Kumagai et al., 1999). Consequently, Cdc7p has been referred to as the DDK (Dbf4-dependent kinase; Nasmyth, 1996b). In all cases, Dbf4p is an unstable protein whose levels peak at the time of Cdc7p activation at the G1-S phase boundary. In budding yeast, DBF4 mRNA levels are high during G1 phase, but the protein levels are low. The APC (anaphase promoting complex (Zachariae and Nasmyth, 1999) is responsible for degradation of Dbf4p in G1 phase of the yeast cell cycle. The APC is also responsible for degradation of mitotic cyclins as cells exit anaphase. Therefore, both CDKs and the Cdc7p kinase become inactive as cells enter G1 phase (Fig. 1).

Dbf4p levels are also regulated by the Rad53p protein kinase (Dohrmann et al., 1999; also known as Chk2 and Cds1; for review, see Weinert, 1998). Rad53p is needed for the DNA checkpoint, which is a surveillance mechanism to monitor DNA replication and DNA damage (Weinert, 1998). As part of this checkpoint, Rad53p prevents cells from entering mitosis if the DNA is damaged or DNA replication is blocked. Consequently, rad53 mutant cells die when the DNA is damaged or replication is blocked, because they enter mitosis with damaged DNA or unreplicated DNA, respectively. Unlike other checkpoint proteins, Rad53p also has an essential function, which might be the regulation of deoxyribonucleotide (dNTPs) precursors. This idea is based on results in which levels of dNTPs elevated either by a sml1 mutation or by overexpression of ribonucleotide reductase can replace or bypass the essential function of Rad53p but not the checkpoint function (Huang et al., 1998; Zhao et al., 1998). My laboratory isolated a rad53-31 mutation, which is lethal in combination with cdc7 mutations, and showed that loss of Rad53p reduces both Dbf4p and mRNA levels (Dohrmann et al., 1999). However, the rad53-31 mutant retains checkpoint function. Thus, regulation of Dbf4p is part of the essential nature of Rad53 in budding yeast and does not appear to involve the DNA checkpoint. We hypothesize that Rad53p binds to and stabilizes Dbf4p, perhaps by phosphorylating it. The stabilized Dbf4p would then increase its own mRNA levels as part of a positive feedback loop. Obviously, stabilization of Dbf4p levels by Rad53p is important but is not essential, given that deletion mutants of rad53 are viable when bypassed by elevated levels of dNTPs. Control experiments have shown that elevating dNTPs by itself has no effect on Dbf4p levels (Dohrmann et al., 1999).

Neither Cdc7p nor Dbf4p plays a part in DNA-checkpoint control in most strains of budding yeast, and most checkpoint mutants, such as rad53, rad9 or mec1, do not affect cell cycle arrest in cdc7 or dbf4 mutants (Weinert et al., 1994). However, some cdc7 or dbf4 budding yeast strains have a defective DNA checkpoint (see above). This defect is attributable to strain background (Toyn et al., 1995). In fission yeast, the homologues of CDC7 and DBF4 are part of this DNA checkpoint. In fission yeast strains in which they are mutated, DNA replication does not occur and yet the cells still divide as they are checkpoint defective (Brown and Kelly, 1999; Masai et al., 1995; Takeda et al., 1999). The Dbf4p homologue, Dfp1p is phosphorylated in a Rad53-dependent manner when cells are blocked in S phase (Brown and Kelly, 1999; Masai et al., 1995; Takeda et al., 1999). In the mold Aspergillus, the Dbf4p homologue is called NimOp (James et al., 1999). nimOts mutants also exhibit defective DNA replication and lack DNA-checkpoint control. Therefore, in some eukaryotic cells, Cdc7p-Dbf4p might be part of a DNA-checkpoint response in which Rad53p phosphorylates Dbf4p. Rad53p might normally be responsible for stabilizing Dbf4 protein levels, but when the DNA checkpoint is activated, increased phosphorylation of Dbf4p by activated Rad53p could be inhibitory. In this way, differential phosphorylation of Dbf4p could explain how Rad53p both positively and negatively regulates Cdc7p-Dbf4p action.

The 58-kDa Cdc7p is conserved to a greater degree than is the 80-kDa Dbf4p (Table 1). This has made discovery of Dbf4 homologues by protein sequence comparison, which had been successful for identifying homologues of Cdc7 (Hess et al., 1998; Jiang and Hunter, 1997; Masai et al., 1995; Roberts et al., 1999; Sato et al., 1997), more difficult. Fortunately, Dbf4 has been cloned by a yeast two-hybrid approach (Jackson et al., 1993) from fission yeast and humans (Jiang et al., 1999; Takeda et al., 1999; R. Hollingsworth, personal communication).

Other Dbf4p homologues might exist and function in different developmental contexts. The Drosophila DBF4 homologue is called chiffon and was isolated as a female sterile mutant because of a defect in egg chorion gene amplification (Landis and Tower, 1999). Mutants in the ORC (origin recognition complex), a conserved multiprotein complex that binds to replication origins (see below), also have this phenotype (Austin et al., 1999; Landis et al., 1997). Unlike yeast DBF4, Drosophila chiffon is not an essential gene. Fission yeast have at least two homologues: dfp1+ (also called rad35+ or him1+), which is needed for mitotic DNA replication; and Srad35 (similar to rad35+), which was discovered by the S. pombe genome project and whose function is unknown (Landis and Tower, 1999). The hypothesis that a variety of Dbf4p proteins activate Cdc7p under different conditions (Landis and Tower, 1999) is similar to that involving activation of CDKs by different cyclins (Sherr and Roberts, 1999). Note that different MCM proteins, which are the substrates for Cdc7p-Dbf4p (see below), are expressed during Xenopus development (Sible et al., 1998). Therefore, different Cdc7p partners might be expressed during development and cause Cdc7p to phosphorylate different substrates.

Substrates

Initial insight into the physiological substrates of Cdc7p-Dbf4p came from genetic and molecular studies in yeast. Earlier studies had used calf-thymus histone H1 as a substrate for Cdc7p-Dbf4p (Hollingsworth and Sclafani, 1990; Yoon and Campbell, 1991). However, H1 is an inefficient and obviously a nonphysiological substrate (Oshiro et al., 1999; Shellman, 1997). Genetic studies have shown that compensatory mutations in dbf4 suppress a mutation in MCM2 (Lei et al., 1997), which, like MCM5, encodes a component of the MCM complex found at chromosomal origins of DNA replication. In fact, the otherwise essential CDC7 and DBF4 genes can be deleted in the presence of the mcm5-bob1 mutation (Hardy et al., 1997). The bob1 mutation in MCM5 changes a conserved proline residue to leucine (P83L), which alleviates the need for the function of either Cdc7p or Dbf4p. The bob1 mutation therefore bypasses CDC7 and DBF4. In contrast, the bob1 mutation cannot bypass other genes required for DNA replication, including those that encode all three DNA polymerases, cdc45 or any other MCM genes (Hardy et al., 1997; Jackson et al., 1993; Pahl, 1994; R. A. Sclafani, unpublished). To determine whether bob1 can bypass CDK-Clb function in DNA replication, we used a cdc4 mutation. This was because there are six different CLBs, any one of which can be used in DNA replication (Nasmyth, 1996a). In the absence of Cdc4p, all CDK-Clb activity is inhibited by Sic1p, a CDK inhibitor, and the cells arrest at the G1-S boundary (Fig. 1; Schwob et al., 1994). The bob1 mutation is unable to bypass the cdc4 block and, hence, is also unable to bypass the role of CDK-Clb in replication (Hardy et al., 1997; R. A. Sclafani, unpublished). On the basis of the above studies, I propose that the MCM complex of proteins is the only essential substrate of Cdc7p-Dbf4p for DNA replication. In this scenario, phosphorylation of the MCM complex by Cdc7p kinase is the only step bypassed in bob1 mutants.

The MCM complex is a hexamer of six different proteins, Mcm2-Mcm7. MCM2-MCM7 are members of a gene family conserved in Archaea and eukaryotes from yeast to humans that share homology with ATPases and DNA helicases (for reviews, see Kearsey and Labib, 1998; Tye, 1999). The MCM complex might be the initiating DNA helicase during DNA replication, functioning similarly to E. coli dnaBp or SV40 T antigen. Recently, potent DNA helicase and ATPase activity of a recombinant archaeal MCM protein has been demonstrated (Chong et al., 2000; Kelman et al., 1999). The current model for the initiation of DNA replication (Fig. 2) in eukaryotes has the MCM complex hexamer loaded onto the ORCp bound to the origin (see reviews by Dutta and Bell, 1997; Tye, 1999). The ORCp recognizes the DNA sequence of the origin and is in turn recognized by Cdc6p. DNA replication occurs by combined action of both CDK-Clb and Cdc7p-Dbf4p on one or more members of this pre-replication complex. Few changes occur in the origin DNA footprint that is produced by the bound pre-replication complex when only CDK-Clb, and not Cdc7p-Dbf4p, is active (Brown et al., 1991; Diffley et al., 1994). When both kinases become active, the pre-replication complex changes to the post-replication complex, which produces a smaller DNA footprint that is similar to that of ORC bound to the origin in vitro (Diffley et al., 1994). DNA replication also cannot occur in G1 phase if only Cdc7p-Dbf4p but not CDK-Clb is activated (Oshiro et al., 1999). Dbf4p binds to an origin in a one-hybrid assay (Dowell et al., 1994), and recent molecular data show that both Cdc7p and Dbf4p are bound to chromatin (Pasero et al., 1999; Weinreich and Stillman, 1999). Together, these results support a model in which Cdc7p-Dbf4p is targeted to the pre-replication complex by Dbf4p at origins to catalyze the initiation of DNA synthesis. A large number of molecular studies point to the MCM complex as a substrate for Cdc7p-Dbf4p (Brown and Kelly, 1998; Jiang et al., 1999; Lei et al., 1997; Oshiro et al., 1999; Roberts et al., 1999; Sato et al., 1997; Weinreich and Stillman, 1999). Discrepancies exist concerning which of the six members of the MCM complex can be phosphorylated by Cdc7p. However, all the studies agree on two points: Mcm2p is a good substrate, and Mcm5p is a poor one. Several in vivo studies point to Mcm2p as the substrate. Mcm2p changes its mobility on 2D-protein gels to a less phosphorylated, or less acidic, form in a dbf4ts mutant (Lei et al., 1997). Phosphopeptides from cellular MCM2 co-migrate with those of MCM2 phosphorylated in vitro by recombinant human CDC7-DBF4 (Jiang et al., 1999). When presented with the entire six-member MCM complex, only Mcm2 is phosphorylated by purified Cdc7p-Dbf4p from fission yeast (Brown and Kelly, 1998) and by CDC7-DBF4 from human cells in vitro (Jiang et al., 1999). Some in vitro studies have localized the phosphorylation site to the N terminus of Mcm2 (Jiang et al., 1999; Oshiro et al., 1999; Weinreich and Stillman, 1999). Only the unphosphorylated MCM complex can bind to Cdc7p, given that the two proteins are bound to each other only during interphase and not during mitosis in frog (Roberts et al., 1999). Interestingly, it is the Mcm5 subunit in which the bob1 P83L mutation can produce a bypass of Cdc7p and Dbf4p (Hardy et al., 1997). This proline residue is conserved in Mcm2p, Mcm4p and Mcm5p. In contrast, a similar proline-to-leucine change in either Mcm2 or Mcm4 does not bypass the requirement for Cdc7p (L. Pessoa-Brandão and R. A. Sclafani, unpublished). This suggests that Mcm5 has a role different from that of Mcm2 or Mcm4. Both DNA helicase and ATPase activity has been shown for human MCM4-MCM6-MCM7 sub-complexes (Ishimi, 1997; You et al., 1999). Consistent with the different roles of MCM proteins is the fact that mutations in different MCM proteins result in differential effects on DNA helicase and ATPase activity (You et al., 1999). Some multimeric DNA helicases display negative cooperativity for ATP and DNA binding, which supports a ‘rolling’ model of unwinding (Lohman and Bjornson, 1996). I propose an allosteric model (Fig. 2), in which phosphorylation of Mcm2p by Cdc7p-Dbf4p confers an allosteric change on Mcm5p and thus produces an active, ‘rolling’ MCM helicase. Mcm5p and Mcm2p might act as inhibitors of helicase activity (Hardy et al., 1997; Stillman, 1994) in a similar way to that in which lambda P protein inhibits the dnaB helicase during coliphage lambda DNA replication (Alfano and McMacken, 1989). Phosphorylation of Mcm2p by Cdc7p and a conformational change in Mcm5p might then allow the MCM replicative helicase to move with the replication fork. The finding that the helicase activity of human MCM4-MCM6-MCM7 complexes can be inhibited by addition of the MCM2 protein is consistent with this ‘inhibitor’ model (You et al., 1999).

Cdc7p is better understood than most other protein kinases: upstream regulatory events and downstream substrates have been clearly identified. Future studies will focus on precise determination of the effect of phosphorylation of the MCM complex by Cdc7p on the function of the complex at origins of DNA replication. These studies must include an analysis of the function of each individual MCM subunit in DNA replication, as well as of interactions with other members of the DNA replication complex, including Cdc6, RFA, RFC and DNA polymerases. Furthermore, the ill-defined positive role of CDKs in DNA replication (Nasmyth, 1996a) must be determined, given that both kinases are clearly required for the initiation of DNA replication. The fact that Cdc7p-Dbf4p is evolutionarily conserved will be helpful in that in vitro frog (Walter et al., 1998), human (Stoeber et al., 1998) and yeast (Pasero and Gasser, 1998) DNA replication systems can be used. An additional challenge will be to identify Cdc7p substrates in meiosis and DNA repair. These studies will have important clinical value, because both Cdc7p-Dbf4p (see above) and the MCM complex might be good biomarkers for neoplastic cells (Stoeber et al., 1999; Williams et al., 1998).

Support for writing this review comes from a PHS grants awarded to R.A.S. (GM35078) and to Paul Bunn, Jr (CA58187). I thank all those who sent me their manuscripts, reprints and personal communications. I also thank Paul Dohrmann, Luis Pessoa-Brandão, and Angela Pierce for helpful comments on the manuscript.

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