Identification of Orthologs of Dbf4 in Other Species

Purification of Hsk1 kinase from fission yeast led to identification of Dfp1, the fission yeast ortholog of Dbf4 (Brown and Kelly 1998). Two-hybrid screening with Hsk1 as a bait also led to the identification of Him1, identical to Dfp1. Interestingly, dfp1/ him1 was found to be allelic to rad35, a radiation-sensitive mutant, implicating Dbf4 in DNA damage response pathway (Takeda et al. 1999). In Aspergillus, a mutant called nimO was isolated and was shown to encode a Dbf4 homologue (James et al. 1999). Human homologue of Dbf4, ASK (activator of S phase kinase), was isolated by two-hybrid screening using huCdc7 as a bait (Kumagai et al. 1999; Jiang et al. 1999).

A second Dbf4 subunit in human was identified and named Drf1 or ASKL1 (Montagnoli et al. 2002; Yoshizawa-Sugata et al. 2005). Drf1/ASKL1 can bind and activate huCdc7, but its role during cell cycle in human cell lines may be secondary. In fission yeast, a second set of Cdc7-Dbf4, Spo4-Spo6, is present which functions specifically during the sporulation/second meiotic division stage of meiosis (Nakamura et al. 2000). No functional homologues of Spo4-Spo6 have been identified in other species.

Activation of Cdc7 Kinase by Dbf4 Protein

Analysis of sequences of Dbf4 and its orthologs revealed the presence of three conserved motifs, motif-N, motif-M, and motif-C (Masai and Arai 2000; Fig. 1). Further analyses indicated that motif-M and motif-C can bind to Cdc7 independently. Binding of both motifs to Cdc7 is required for full activation of Cdc7 kinase. The sequences and length connecting motif-M and motif-C can be varied without affecting the kinase activity, suggesting that they serve as a linker sequence (Ogino et al. 2001; Kitamura et al. 2011). Motif-M is a proline-rich motif with no apparent similarity to known motifs. Motif-C is a C₂H₂-type zinc finger-like structure. Motif-N is composed of a single copy of a BRCT-like sequence, which could be uniquely replaced by the BRCT motif from Rev1 (Harkins et al. 2009). It was reported that zinc finger mutation exhibited slowed S-phase, DNA damage sensitivity, and hypo-mutagenic phenotype following UV irradiation. This mutant does not interact with Mcm2 and exhibits reduced phosphorylation. The same mutant was sensitive to long-term exposure to HU or MMS (Jones et al. 2010). Motif-M alone can sustain mitotic viability (Fung et al. 2002), but both motif-M and motif-C are required for viability through meiosis in fission yeast (Ogino et al. 2001). On the other hand, motif-N is not essential for viability, although it is required for interaction with Rad53 checkpoint kinase and is involved in conferring resistance to genotoxic agents and in checkpoint responses (Chen et al. 2013; Matthews et al. 2014). In mammalian cells, motif-M and motif-C are sufficient for maximum kinase activity, but motif-N is also required for viability (Yamashita et al. 2005).

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There is a long C-terminal tail segment in Dbf4/ASK from higher eukaryotes, which is not conserved except for the very C-terminal sequences rich in serines and threonines. They are major autophosphorylation sites. Truncation of the C-terminal 50 amino acids of human ASK was shown to hyperactivate the Cdc7 kinase, suggesting autoinhibition of Cdc7-ASK kinase by the C-terminal tail. LEDGF protein, a co-factor of human immunodeficiency virus DNA integration, interacts with this segment and relieves the autoinhibition (Hughes et al. 2010).

Functions of Dbf4/ASK During Cell Growth

A temperature-sensitive mutant of dbf4 in budding yeast ceases growth at the onset of S phase, an identical position with that of cdc7(ts) cells. The arrested cells can reversibly enter the S phase upon return to a permissive temperature. Analyses of a temperature-sensitive mutant of nimO, the Aspergillus homologue of Dbf4, showed that NimO is required for initiation of DNA synthesis and for efficient progression through S phase as well as for DNA replication checkpoint coupling S and M phases (James et al. 1999). Drosophila homologue of Dbf4, chiffon, is required for chorion gene amplification. Hypomorphic mutant alleles of the chiffon gene cause thin, fragile chorions and female sterility. Null alleles of chiffon had the additional phenotypes of rough eyes and thin thoracic bristles, phenotypes often associated with disruption of normal cell cycle progression (Landis and Tower 1999).

Dbf4 and Dfp1/Him1 are hyperphosphorylated in response to replication stress (such as treatment with hydroxyurea, which inhibits cellular nucleotide reductase and depletes cellular nucleotide pool). This phosphorylation of Dbf4 or Dfp1/Him1 depends on both Cdc7/Hsk1 and checkpoint kinase Rad53 or Cds1, respectively (Takeda et al. 2001; Brown and Kelly 1999). It has been proposed that this phosphorylation somehow inhibits the function of Dbf4, which contributes to suppression of firing of late origins. However, the nature of this inhibition is still unknown. Extensive mutagenesis of potential phosphorylation sites on Dbf4 rendered the mutant Dbf4 refractory to the checkpoint inhibition, and combination of the Dbf4 mutant with a similar phosphorylation site mutant of Sld3 abrogated the checkpoint inhibition of late origin firing, showing that Dbf4 is a critical target of DNA replication checkpoint (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). Human Dbf4/ASK and fission yeast Hsk1 may also be hyperphosphorylated by replication stress (Heffernan et al. 2007; Snaith et al. 2000). Dbf4 is generally limiting in quantity, and overexpression of Dbf4 can cause late-firing origins to fire earlier in S phase (Mantiero et al. 2011). Similar findings were made also in fission yeast (Patel et al. 2008; Wu and Nurse 2009).

In mouse ES cells, conditional knockout of Dbf4/ASK is lethal and cells undergo cell death (Yamashita et al. 2005), as was observed in ES cells in which the Cdc7 gene was conditionally knocked down.

Functions During Meiosis

Initial characterization of budding yeast cdc7(ts) indicated the essential role of Cdc7 for meiotic recombination, but not for premeiotic DNA replication. In fission yeast as well, cells are arrested with one nucleus in an hsk1(ts) cells. In both fission yeast and budding yeast, initiation of meiotic recombination, i.e., induction of DSB (double-stranded DNA breaks), does not occur in the absence of Hsk1/Cdc7 (Ogino et al. 2006). Furthermore, Mer2, a factor essential for loading of DSB endonuclease Spo11, was identified to be a critical target of Cdc7 (Sasanuma et al. 2008; Wan et al. 2008). Premeiotic DNA replication can proceed albeit at slightly slower rate in hsk1(ts) mutant or cdc7 as (a shorkat form of Cdc7 that can be chemically inactivated) in which Cdc7 can be chemically inactivated (Ogino et al. 2006; Lo et al. 2008).

On the other hand, repression of dbf4⁺ expression regulated by the tet promoter suppressed premeiotic DNA replication in budding yeast when it was suppressed before meiosis was induced, but DNA replication was observed if dbf4⁺ expression was suppressed later, presumably due to inadequate suppression of dbf4⁺ expression. This result suggests that Dbf4 function is required for premeiotic DNA replication. In the latter cells, premeiotic S phase was completed, but the meiosis was still arrested before anaphase I. This arrest was relieved by rec8 deletion, suggesting a crucial role of Cdc7 during meiotic chromosome segregation (Valentin et al. 2006). Indeed, Rec8 cleavage by separase is regulated by phosphorylation mediated by Cdc7 or casein kinase 1 (Katis et al. 2010). Cdc7 also regulates monopolar attachment of sister kinetochores by regulating the recruitment of the monopolin complex to kinetochores through phosphorylation of monopolin subunit Lrs4 (Matos et al. 2008). Thus, Dbf4, in a complex with Cdc7, may regulate multiple steps of meiotic cell cycle.

Other Dbf4-Related Molecules

Another Cdc7-Dbf4-related complex, the Spo4(Cdc7-like)-Spo6(Dbf4-like) complex, is expressed in fission yeast during late meiosis and is specifically required for the sporulation stage (Nakamura et al. 2000). Kinase complexes related to Spo4-Spo6 have not been found in other species.

A second Dbf4/ASK-like molecule, Drf1/ASKL1, was identified in silico on the human genome and was shown to function as an activation subunit for human Cdc7 kinase (Montagnoli et al. 2002; Yoshizawa-Sugata et al. 2005). The expression level of Drf1/ASKL1 increases during late S/G2, and inhibition of its expression resulted in accumulation of late S-G2/M populations. In contrast to Dbf4/ASK, Drf1/ASKL1 is present mostly in the nuclear soluble fractions, not in chromatin-bound fractions. Drf1/ASKL1 has been identified only in human and Xenopus.

Association of Dbf4 with Origin Sequences in Budding Yeast

One-hybrid assays showed that Dbf4 interacts with the replication origin sequences in budding yeast (Dowell et al. 1994). Mapping of the interacting segment on Dbf4 indicated motif-N as the origin-interacting domain. Thus, Dbf4 targets Cdc7 kinase at the origin of DNA replication. At the origin, Cdc7 targets pre-replicative complex (pre-RC) which is generated on chromatin during early G1. Among the components of pre-RC, MCM is the critical and conserved substrate of Cdc7 (Lei et al. 1997; Sato et al. 1997). See the section of “Cdc7” for details on how Cdc7-mediated phosphorylation may activate initiation of DNA replication.

Interaction of Dbf4 with Other Replication Factors

Yeast two-hybrid analyses indicated the interaction of mouse Dbf4/ASK with Orc1, Orc2, Orc5, and Orc6 as well as with MCM2, MCM3, MCM4, and MCM7, consistent with the interaction of Dbf4 with pre-RC assembled at origins in budding yeast (Kneissl et al. 2003). In the same report, mouse Cdc7 was reported to interact with Orc1 and Orc6 and with MCM2, MCM4, MCM5, and MCM7 in two-hybrid assays. Two-hybrid assays also indicate that budding yeast Dbf4 interacted most strongly with Mcm2, whereas Cdc7 associated with both Mcm4 and Mcm5. They found most strong interaction between Dbf4 and N-terminal segment of Mcm2, which may serve as a second Cdc7 docking site in addition to that found in Mcm4 (Sheu and Stillman 2006).

Regulation of Expression of Dbf4 During the Cell Cycle

Expression of Dbf4 is cell cycle regulated. Regulation is generally on both transcription and protein levels. In budding yeast, transcription of Dbf4 is regulated during cell cycle, peaking during G1 (Chapman and Johnston 1989). The promoter contains a MluI cell cycle box (or MCB) and may be regulated by the MCB-binding factor (MBF). Furthermore, the budding yeast Dbf4 protein is degraded by APC during G1 phase. The protein is present during S through G2 phase, coinciding with the active Cdc7-Dbf4 kinase activity during this period. The potential degradation signal was identified in the N-terminal segment of Dbf4 (Oshiro et al. 1999; Ferreira et al. 2000). Similar regulation is likely to operate for the fission yeast Dfp1/Him1 gene, which is expressed specifically during S through G2/M phase (Brown and Kelly 1998; Takeda et al. 1999).

Expression of mammalian Dbf4/ASK is also cell cycle regulated. The transcript is repressed in the quiescent cells and induced after growth stimulation. A 63-base pair ASK promoter segment was identified, which is sufficient for mediating growth stimulation (Yamada et al. 2002). This minimal promoter segment contains an Sp1 site but no canonical E2F site but can be activated by ectopic E2F expression. Within the 63-base pair region, the Sp1 site and other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Another report proposed the presence of MCB in the core promoter region of human Dbf4/ASK (Wu and Lee 2002). The Dbf4/ASK protein levels decrease during G1 phase, and a part of this may be attributed to cell cycle-dependent protein degradation.

Developmental Role of Dbf4

A role of Dbf4 in heart/eye development was suggested in Xenopus (Brott and Sokol 2005). Dbf4/ASK inhibits the canonical Wnt signaling pathway, possibly through interacting with Frodo. This role of Dbf4 does not involve its ability to activate Cdc7 kinase, since the Dbf4-motif-M, which is known to be essential for Cdc7 kinase activation, is not required for its role in heart development. Expression of Drf1/ASKL1 and Dbf4/ASK molecules is developmentally regulated in Xenopus. Drf1/ASKL1 is predominantly expressed in early development but is later replaced by Dbf4/ASK in somatic cells (Takahashi and Walter 2005).

Those who are interested in learning more about eukaryotic DNA replication are recommended to read the reference (Masai et al. 2010).