Historical Background
Cdc7 was originally discovered as a temperature-sensitive mutant of budding yeast defective in progression of cell cycle (Hartwell 1973). The growth of cdc7(ts) is arrested immediately before the onset of the S phase at a nonpermissive temperature. Upon return to a permissive temperature, cells resume growth and can complete S phase in the absence of ongoing protein synthesis. This led to the notion that Cdc7 is required for DNA replication at the stage where all other proteins required for DNA synthesis are prepared. Cdc7 was later cloned and was identified as encoding a serine/threonine kinase (Hollingsworth and Sclafani 1990). Dbf4, another cell cycle regulator required at the onset of the S phase, was then shown to encode an activation subunit for Cdc7. An ortholog of Cdc7 was first identified in fission yeast (Hsk1; (Masai et al. 1995)) and was later shown to be conserved in higher eukaryotes as well (Sato et al. 1997; Jiang et al. 1997).
Structure of Cdc7 Kinase and Activation by Dbf4 Subunit
Cdc7 belongs to a rather unique branch of the kinase phylogeny tree, and the closest member may be the casein kinase. Cdc7 kinase is unique in that it carries two or three so-called kinase insert sequences at the conserved locations within the kinase-conserved domains (Kim et al. 1998). The sequences and lengths of the kinase insert are not generally conserved. Analyses in human Cdc7 kinase indicated that the kinase insert II between the kinase domains VII and VIII is required for efficient nuclear localization as well as for chromatin association of Cdc7 (Kim et al. 1998; Masai and Arai 2002), whereas a small segment (DAM-1, Dbf4/ASK association motif-1) in the kinase insert III between the domains X and XI is required for interaction with Dbf4/ASK activation subunit. A conserved sequence present at the C-terminus of Cdc7 (DAM-2) also plays an essential role in interaction with Dbf4/ASK. Furthermore, DAM-1 and DAM-2 were shown to interact with motif-M and motif-C of Dbf4/ASK (Kitamura et al. 2011), respectively. Dbf4/ASK stimulates binding of ATP to the Cdc7 catalytic subunit as well as recognition of specific substrates ((Ogino et al. 2001; Kitamura et al. 2011); Fig. 1). Recently, the structure of Cdc7-ASK complex was determined by X-ray crystallography (Hughes et al. 2012).
A proposed model for interaction of Cdc7 kinase with Dbf4/ASK subunit. Upper: Schematic drawing of overall structure of human Cdc7. Two small segments of Cdc7 (pink boxes) termed DAM-1 and DAM-2 interact with motif-M and motif-C, respectively, of Dbf4/ASK protein (Ogino et al. 2001; Kitamura et al. 2011). Lower: Schematic drawing showing interaction between Cdc7 and Dbf4/ASK. The interaction induces conformational change in Cdc7, thus permitting access to ATP and facilitating the recognition of substrates
Identification of Orthologs of Cdc7 in Other Species
An ortholog of budding yeast Cdc7 was first identified in fission yeast (hsk1+; (Masai et al. 1995)). Hsk1 and Cdc7 share about 40% identity in the kinase-conserved domains (Kim et al. 1998). Hsk1 contains characteristic insert sequences at the conserved locations among the kinase-conserved domains, as stated above. Cdc7 homologues were then identified in humans, mice, drosophila, and Xenopus (Jiang and Hunter 1997; Sato et al. 1997; Kim et al. 1998; Stephenson et al. 2015). It is now known to be conserved over a wide range of species (Masai and Arai 2002). In fission yeast, another kinase, spo4+, related to Cdc7, was identified (Nakamura et al. 2002). Spo4 is expressed specifically during late meiosis and forms a complex with Spo6, a meiosis-specific Dbf4-like molecule. Spo4-Spo6 is required for second meiotic cell division and sporulation.
Regulation of Expression
In normal proliferating yeast cells, Cdc7 protein is expressed constitutively during cell cycle. In fission yeast, the transcription of hsk1+ slightly oscillates during cell cycle, peaking at the G1/S boundary. However, the protein level of Hsk1 is more or less constant during cell cycle (Takeda et al. 1999).
Expression levels of budding yeast Dbf4 and fission yeast Dfp1/Him1 oscillate during cell cycle. MluI box protein or Cdc10 transcription factor regulate their transcription. Cdc7 kinase activity increases during S phase and is retained until the end of M phase. This regulation largely depends on the level of Dbf4 activation subunit. In budding yeast, Dbf4 possesses a degradation box, which triggers its APC-dependent degradation during G1 phase (Oshiro et al. 1999; Ferreira et al. 2000).
In cycling cells, human Cdc7 protein level decreases during G1 phase, probably due to proteolytic degradation, since the Cdc7 protein expressed from a constitutive promoter similarly oscillates during cell cycle (Masai et al. 2006).
The transcriptions of Cdc7 and Dbf4/ASK in mammals are repressed during the resting state (Kumagai et al. 1999). Upon growth resumption after addition of serum, they are derepressed (Kim et al. 1998). A 231 bp segment of the muCdc7 promoter containing three E2F binding sites and a Sp1 site but lacking a TATA sequence is sufficient for response to growth stimulation. On the other hand, a 63 bp segment from the human Dbf4/ASK promoter contains Sp1 and E2F binding sites and can constitute an active promoter segment that can respond to growth factors. Inhibitory E2F (E2F4 or E2F5) binds to the promoter during the resting state and is replaced by activating E2F upon growth restimulation (Yamada et al. 2002; Wu and Lee 2002).
Cdc7 protein associates with chromatin during S phase and dissociates from it during G2/M phase (Masai et al. 2006). It is expressed also during M phase and is present in cytoplasm. It is phosphorylated by Cdk (cyclin-dependent kinase) during M phase, and the phosphorylated Cdc7 is dissociated from chromatin, although the significance of this phosphorylation is not clear at the moment.
Substrate Specificity of Cdc7 Kinase
Cdc7 is a serine-threonine kinase, the primary structure of which shares some similarity with casein kinase. Like casein kinase, Cdc7 is an acidophilic kinase, favoring the acidic environment surrounding the target site. It has been reported that prior phosphorylation of a target protein by another kinase such as Cdk or a checkpoint kinase can stimulate Cdc7-mediated phosphorylation by creating acidic environment surrounding the target sites (Masai et al. 2000; Montagnoli et al. 2006; Wan et al. 2008; Randell et al. 2010). SSP or STP may be one of the typical Cdc7 target sequences in which second serine or threonine is first phosphorylated by Cdk and the first serine is subsequently phosphorylated by Cdc7 (Cho et al. 2006; Masai et al. 2000; Montagnoli et al. 2006; Wan et al. 2008).
Cdc7 kinase preferentially targets chromatin-associated Mcm2-7 complexes that is tightly linked to the origin DNA (Francis et al. 2009). Efficient targeting and phosphorylation of Mcm on the chromatin may be related to the presence of histone, since Cdc7-Dbf4 is highly stimulated by the acidic compounds including spermine, spermidine, and histones (Kakusho et al. 2008).
Recent reports indicate the presence of substrate recruiter proteins for Cdc7 kinase. DDD (DDK [Dbf4-dependent kinase] docking domain; aa175–333) was identified in budding yeast Mcm4, which facilitates the Cdc7-mediated phosphorylation at the adjacent N-terminal segment of Mcm4 (Sheu and Stillman 2006). During the replication-coupled meiotic recombination, Tof1-Csm3 (Tim-Tipin in mammals) complex at the replication fork recruits Cdc7 kinase for phosphorylation of Mer2, a protein crucial for DSB formation (Murakami and Keeney 2014). More recently, human Claspin was shown to possess the segment named AP (acidic patch) to which Cdc7 binds. The recruitment of Cdc7 to AP of Claspin plays an important role in efficient phosphorylation of Mcm required for initiation in normal cells as well as in phosphorylation of Claspin itself (Yang et al. 2016). Similarly, the fission yeast Mrc1, the counterpart of Claspin, also has a segment named HBS that recruits Cdc7 kinase, and HBS is important for regulated firing of early-firing origins on the fission yeast chromosomes (Matsumoto et al. 2017).
Functions in Initiation of DNA Replication
Cdc7 is essential for cell viability in yeasts under normal growth condition. Conditional knockout of Cdc7 gene in mouse embryonic stem (ES) cells resulted in inhibition of DNA synthesis and eventual p53-dependent cell death due to accumulation of DNA damages (Kim et al. 2002), indicating that Cdc7 is also essential for mammalian cell growth. Early characterization of cdc7(ts) in budding yeast suggested its essential role in initiation of S phase. Later, it was shown that Cdc7 is required for initiation of DNA replication at each replication origin throughout S phase (Donaldson et al. 1998; Bousset and Diffley 1998). One-hybrid assays showed association of Dbf4 with replication origins in budding yeast (Dowell et al. 1994). These findings led to the notion that the pre-RC generated at each origin may be the target of Cdc7. Indeed, human Cdc7 was shown to phosphorylate MCM (minichromosome maintenance) in vitro (Sato et al. 1997). In budding yeast, MCM2 was shown to be phosphorylated in a manner dependent on Cdc7 (Lei et al. 1997). Accumulating evidence pointed to MCM complex as a major conserved substrate of Cdc7 kinase. Phosphorylation of MCM2, MCM4, and Mcm6 at the N-terminal segments promotes association of Cdc45 with pre-RC (pre-replicative complex) ((Masai et al. 2006; Sheu and Stillman 2006); Figs. 2, 3 and Table 1), facilitating generation of the replication fork complex containing an active DNA helicase. In fission yeast, Hsk1 was shown to be essential for loading of Sld3 which is needed for assembly of an active replication fork. Hsk1 was also shown to regulate the efficiency of origin firing in fission yeast (Patel et al. 2008; Wu and Nurse 2009). It was also shown in yeast that increased Cdc7 kinase activity (as well as overexpression of limiting Sld3, Sld2, Dpb11, or Cdc45) is sufficient to permit late origins to fire early (Mantiero et al. 2011; Tanaka et al. 2011).
Structure of Cdc7 as revealed by X-ray crystallography (Adapted from Matthews and Guarne 2013)
Roles of Cdc7 kinase in initiation of DNA replication. Cdc7-Dbf4 kinase recognizes pre-replicative complex (pre-RC) on the chromosome and phosphorylates MCM complex. This will facilitate the recruitment of replisome factors including Cdc45 required for generation of an active replication fork
The roles of Cdc7-mediated phosphorylation of Mcm have been a focus of intense studies. It was reported that Cdc7-mediated phosphorylation of Mcm2 results in weakened interaction between Mcm2 and Mcm5, causing ring opening of Mcm2~7, resulting in extrusion of single-stranded DNA (Bruck and Kaplan 2015).
Recently, using the in vitro replication system reconstituted with purified components, it was shown that Cdc7 promotes association of Sld3 with the phosphorylated residues on Mcm4 and Mcm6. Sld3 then recruits Cdc45 to the preRC, leading to generation of CMG helicase (Deegan et al. 2016).
Functions During Mitotic Cell Cycle
Although the major and conserved role of Cdc7 kinase is in regulation of initiation of DNA replication, recent investigation has pointed to important roles of Cdc7 kinase in regulation of other mitotic events (see Fig. 4 and Table 1). Centromeric sister chromatid cohesion is partially impaired in hsk1(ts) (Takeda et al. 2001; Bailis et al. 2003). Consistent with this, the mutants are synthetic lethal with Rad21, a cohesin component, and is sensitive to the anti-microtubule drug thiabendazole (TBZ, (Snaith et al. 2000; Takeda et al. 2001)). These results suggest a role of Hsk1 kinase in regulation of sister chromatid cohesion. In Xenopus egg extracts, Cdc7 was shown to form a complex with Scc2-Scc4 which is required for loading of cohesin onto chromatin (Takahashi et al. 2008). The kinase activity of Cdc7 is required for this process, although the exact target of Cdc7 is not known. It is also not known whether Cdc7 behaves similarly in other species including mammals.
Chromosome dynamics regulated by Cdc7 kinase. Various chromosome transactions, as indicated in the figure, are regulated by Cdc7 kinase. The potential target proteins in these events are shown. The red arrows indicate the recruitment by the phosphorylation which has been proven, and blue arrows indicate that which has been speculated but not been proven
Cdc7 kinase accumulates at kinetochores in telophase through the Ctf19 kinetochore complex, recruiting Sld3-Sld7 to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, Cdc7-Dbf4 at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This facilitates robust pericentromeric cohesion in S phase (Natsume et al. 2013). Thus, Cdc7 plays a key role in orchestrating early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.
In budding yeast, Cdc7-Dbf4 interacts with polo kinase (cdc5) through N-terminal nonessential segment of Dbf4 and appears to regulate mitotic exit (Miller et al. 2009; Chen and Weinreich 2010). In fission yeast as well, hsk1(ts) is synthetic lethal with a plo1 (fission yeast homologue of polo kinase) mutant (our unpublished result). In human cancer cells, depletion of Cdc7 inhibited the cell-cycle progression from G2 phase arrest induced by nocodazole (our unpublished result). These results suggest conserved roles of Cdc7 kinase during mitosis.
Functions During Meiotic Cell Cycle
Important roles of Cdc7 in meiotic cell cycle were originally reported in cdc7(ts) mutants of budding yeast (Schild and Byers 1978; see Fig. 4 and Table 1). Defect in meiotic recombination and in synaptonemal complex formation was initially observed, while premeiotic DNA replication was reported not to be significantly affected. More recently, using fission yeast hsk1(ts) mutant, it was demonstrated that Hsk1 is required for formation of DSBs (double-stranded DNA breaks) which is prerequisite for meiotic recombination (Ogino et al. 2006). Premeiotic DNA replication could continue in hsk1(ts) mutant, albeit at a slightly slower rate. Similar conclusion was reached in budding yeast. However, using a tet-repressible transcription system, premeiotic DNA replication was shown to require Dbf4 function in budding yeast (Valentin et al. 2006). Thus, it appears that Cdc7 may facilitate the initiation of premeiotic DNA replication, but its requirement may be less stringent than for mitotic DNA replication. Phosphorylation of Mer2 protein, a component of the Mer2-Mei4-Rec114 complex required for initiation of meiotic recombination, by combined actions of Cdk and Cdc7 was shown to play essential roles in generation of meiotic DSB (Sasanuma et al. 2008; Wan et al. 2008).
Cells arrest with one nucleus state in an hsk1(ts) mutant in meiosis, suggesting that Hsk1 is required also for meiotic cell division. Consistent with this prediction, Cdc7 in budding yeast, in conjunction with casein kinase 1, was shown to phosphorylate Rec8 and to regulate cohesion cleavage by separase. C-terminal truncated form of Dfp1/Him1 (1-519) exhibits defect in meiosis II due to its inability to phosphorylate Rad8, the meiotic homologue of Rad21, and to induce its degradation (Le et al. 2013).
During meiosis I, kinetochores from the sister chromatid are attached by the microtubule from the same spindle pole body (monoorientation). Cdc7 plays an essential role in establishing monoorientation by promoting association of the monopolin complex at kinetochores through phosphorylation of Lrs24, a subunit of the complex, and also through stimulating NDT80 transcription (Lo et al. 2008; Matos et al. 2008).
Cdc7 during meiosis is likely to be conserved in mammalian cells. Cdc7 hypomorphic mutant mice were sterile, and germ cell development was severely disrupted (Kim et al. 2003). Sperm development was arrested at an early prophase, consistent with completion of premeiotic S phase followed by arrest before meiotic cell division.
Functions During Mutagenesis
Roles of Cdc7 in UV-induced mutagenesis were suggested in the initial characterization of budding yeast cdc7(ts) mutants (Njagi and Kilbey 1982a, b; Kilbey 1986). More recently, budding yeast cdc7+ was shown to be required for trans-lesion synthesis branch of Rad6 epistasis group (Pessoa-Brandão and Sclafani 2004; Brandão et al. 2014). In human, replication fork arrest induces Chk1-dependent degradation of Cdh1, leading to inactivation of APC/C(Cdh1). This results in stabilization of Cdc7-ASK on the chromatin, which would interact with Rad18, an E3 ubiquitin ligase, through Dbf4-motif-C. This permits accumulation of Rad18 on the chromatin and recruitment of a lesion-bypass polymerase, Polη (Rad30 counterpart) (Yamada et al. 2013). Another study indicates that serine residues in the C-terminal region of Rad18 near Polη binding segment are phosphorylated by Cdc7, which is required for Polη binding to Rad18 (Day et al. 2010).
Role of Cdc7 Kinase in Replication Checkpoint Regulation
The potential roles of Cdc7 kinase in replication checkpoint response have been controversial. There are two possible ways for involvement in checkpoint responses, one as a target and the other as an activator. The potential roles of Cdc7 in both venues have been investigated.
Induction of DNA damage signals in Xenopus egg extracts led to inhibition of Cdc7 kinase activity and subsequent Cdc45 loading on chromatin that depends on RPA binding to single-stranded DNA regions (Costanzo et al. 2003). It was also reported in budding yeast that replication stress resulted in Rad53-dependent hyperphosphorylation of Dbf4 subunit, leading to reduced Cdc7 kinase activity (Weinreich and Stillman 1999). These results suggest that Cdc7 kinase is one of the replication checkpoint targets. On the other hand, it was also reported that the complex formation, chromatin binding, and kinase activity of the Cdc7-Dbf4 complex are not affected by replication stress in Xenopus egg extracts. This report claimed that Cdc7 promotes replication restart by reducing the checkpoint signal (Tsuji et al. 2008). In human cells, Cdc7 kinase activity was reported not to change after replication stress (Tenca et al. 2007). In fission yeast cells, Hsk1 kinase activity did not change after HU treatment (Matsumoto et al. 2010). Thus, the view that Cdc7 is a target of checkpoint responses is not fully supported by the available biochemical data. However, genetic evidence in yeast strongly indicates that Dbf4 is a critical target of checkpoint response. 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 (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). Thus, checkpoint-induced phosphorylation of Dbf4 somehow inhibits its function, which contributes to suppression of firing of late origins. However, the nature of this inhibition is still unknown.
The evaluation of replication factors as a checkpoint activator is not straightforward, since replication stress checkpoint has been shown to be dependent on the numbers of ongoing replication forks (Shimada et al. 2002; Tercero et al. 2003). Thus, reduced checkpoint reaction by inhibition of a replication factor needs to be carefully evaluated so one can leave out the possibility that it is an indirect effect of reduced DNA replication. Δcdc7 bypassed by mcm4ΔN failed to induce checkpoint in response to replication stress, showing that Cdc7 is required for checkpoint activation (Sheu and Stillman 2010). Similarly, hsk1ts cells is sensitive to HU and HU-induced Mrc1 phosphorylation, and Cds1 activation was severely compromised in this mutant (Takeda et al. 2001). Δhesk1 Δrif1 cells are viable and checkpoint was reduced compared to Δrif1. These results strongly suggest that Cdc7 plays an important role in activation of checkpoint responses. Studies in fission yeast indicated that replication stress-induced Cds1 kinase activation was reduced in a cdc45 mutant but not in mcm or polε mutants. Cdc7 plays a crucial role in loading of Cdc45 onto pre-RC. Therefore, Cdc7-mediated loading of Cdc45 may be somehow linked to activation of checkpoint (Matsumoto et al. 2010). In mammalian cells, Cdc7 knockdown reduced the HU-induced Claspin phosphorylation and checkpoint activation. Like Mrc1, Claspin is phosphorylated by Cdc7 in human cells, and this phosphorylation may play an important role in checkpoint activation (Kim et al. 2008; Rainey et al. 2013). Recent studies indicate that Claspin is an important substrate of Cdc7 during the normal course of DNA replication as well (Yang et al. 2016).
Roles of Cdc7 in Other Chromosome Transactions
Accumulating evidence indicates that Cdc7 regulates diverse chromosome processes (see Fig. 4 and Table 1).
DNA Repair
In fission yeast, Hsk1 phosphorylates Rad9 in the 9-1-1 clamp loader that functions during DNA damage response. This phosphorylation reduces the interaction between 9-1-1 and RPA, resulting in dissociation of Rad9 from the damaged sites and facilitating the repair process (Furuya et al. 2010).
Histone Modification
Replication-associated phosphorylation of threonine 45 of Histone H3 is mediated by Cdc7-Dbf4 in budding yeast and may play a role in maintenance of genomic integrity during DNA replication and repair (Baker et al. 2010). The loss of Histone H3 threonine 45 phosphorylation is associated with replicative defects. Prolonged replication stress induced accumulation of this phosphorylation. This histone modification proceeds in a manner independent of another histone modification, Histone H3 Lys56 acetylation, known to be important for maintenance of genomic integrity during DNA replication and repair. The double mutant defective in both histone modifications is nonviable.
Chromatin Assembly
Human Cdc7 interacts with chromatin assembly factor 1 (CAF1) and phosphorylates p150 subunit. This phosphorylation facilitates interaction of Caf1 with PCNA (proliferating cell nuclear antigen, known as a clamp protein) (Gérard et al. 2006). Thus, Cdc7 may play a role in the coordination between DNA replication and chromatin assembly by Caf1.
Heterochromatin Replication
Although heterochromatin regions are replicated late in the S phase, pericentromeric and mating-type loci are replicated early in fission yeast. This is made possible by selective recruitment of Dpf1/Him1 to Swi1 (HP1 homologue recognizing H3K9methyl) through the PVVTI motif on Dpf1/Him1 (Hayashi et al. 2009).
Hyphal Growth
Cdc7 kinase from Candida albicans, an opportunistic human fungal pathogen, is essential for normal growth, and its repression induced the hyphal growth, suggesting that Cdc7 inhibits the hyphal development (Lai et al. 2016).
Topoisomerase and Centromere
Human Cdc7 interacts with Top2A, a type II topoisomerase, and phosphorylates it during S phase. Dbf4/ASK is localized at centromere during S phase, which promotes recruitment of Top2A to the centromere. The loss of potential Cdc7-mediated phosphorylation sites on Top2A and inhibition of Cdc7 kinase delayed Top2A association with centromere (Wu et al. 2016).
Protein Degradation
Cdc7-mediated stimulation of the protein degradation has been reported. Ams2, a GATA-like transcription factor, regulates S phase-specific expression of histone, and its expression is cell cycle regulated. The protein stability of Ams2 was found to be regulated by phosphorylation of a “phospho-degron” sequence by Hsk1-Dfp1/Him1 (Takayama and Toda 2010). The stability of Mrc1 is also regulated by similar Hsk1-mediated phosphorylation of a degradation box (Shimmoto et al. 2009). Eco1, involved in establishment of cohesion, is sequentially phosphorylated by Cdk1, Cdc7-Dbf4, and Mck1 (a GSK-2 homologue) kinases, which promotes its association with Cdc4 and subsequent degradation (Lyons et al. 2013).
Homologous Recombination
Most recently, it was reported that Cdc7-Dbf4, in conjunction with Cdc5, phosphorylates Mus81-Mms4, a DNA joint molecule resolving nuclease, in an interdependent manner, and Cdc7-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis. Thus, Cdc7 is a novel regulator of homologous recombination (Princz et al. 2017).
Proteins Interacting with Cdc7
Cdc7 is an enzyme which may interact with the substrates only transiently. Thus, interaction with Cdc7-Dbf4 kinase is generally weak. Physical interaction with MCM subunits has been reported, as expected from the fact that MCM is one of the most robust substrates of Cdc7. Yeast two-hybrid assays with mouse Cdc7 showed its interaction with Orc1 and Orc6 and with MCM2, MCM4, MCM5, and MCM7 (Kneissl et al. 2003). In budding yeast, interaction of Cdc7 with other replication factors has been reported. In human cells and fission yeast, Cdc7 (Hsk1) interacted with Claspin (Mrc1) protein (Kim et al. 2008; Uno and Masai 2011). In human cells, Cdc7 interacts with Cdt1, and this interaction may facilitate the loading of Cdc45 onto chromatin (Ballabeni et al. 2009).
Two hybrid assays indicate that 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 (Ramer et al. 2013).
Developmental and Higher-Order Functions of Cdc7
The Drosophila chiffon mutant exhibits thin, fragile chorions and female sterility and is deficient in chorion gene amplification (Landis and Tower 1999). The mutant also shows rough eyes and thin thoracic bristles. Chiffon encodes a Dbf4-like molecule which is required for normal development of Drosophila.
In Xenopus egg extracts, Cdc7-Drf1/ASKL1 is a major Cdc7 complex, but the level of Drf1/ASKL1 decreases and that of Dbf4 increases after gastrulation, when the cell cycle acquires somatic characteristics (Takahashi and Walter 2005). It is not known whether similar developmental regulation operates in human cells.
Xenopus Dbf4 (XDbf4) is required for heart and eye development. XDbf4 inhibits Wnt signaling through interaction with Frodo. This role of XDbf4 is independent of its function as an activator of Cdc7 kinase, since XDbf4 that cannot activate Cdc7 can still rescue the cardiac marker expression induced by XDbf4 depletion (Brott and Sokol 2005).
Cdc7 knockout mice die at E3.5~6.5. Conditional knockout of Cdc7 functions in mouse ES cells results in cessation of DNA replication, accumulation of γH2AX and Rad51 foci, and subsequent p53-dependent cell death. Same treatment in mouse MEF cells caused senescence and did not induce cell death (Kim et al. 2003).
We have developed hypomorphic Cdc7 mice, which show short stature and, most notably, complete defect in germ cell development, and thus are infertile, as stated in an earlier section. Arrest occurred early in meiosis, consistent with the results of fission yeast. The defect in germ cell development was corrected by introducing another copy of Cdc7 gene (Kim et al. 2007).
Cdc7 is expressed in the brain, although the significance of Cdc7 in nonproliferating cells has been unclear. Recently, Cdc7 was reported to be responsible for phosphorylation of Serine 409/Serine 410 in TDP-43 protein in C. elegans. This phosphorylation appears to be conserved also in human. In frontotemporal lobar degeneration (FTLD)-TDP cases, Cdc7 immunostaining overlaps with the phospho-TDP-43 found in frontal cortex. Furthermore, a small molecule Cdc7 inhibitor reduces TDP-43 phosphorylation and prevents neurodegeneration in TDP-43-transgenic animals (Liachko et al. 2013). Genetic analyses of Cdc7 in specific tissues and organs using genetically manipulated mice will shed more light on the physiological roles of Cdc7 in animals.
Is Cdc7 Essential for DNA Replication?
cdc7(ts) mutant initially identified by Hartwell exhibits strong cell cycle arrest at a nonpermissive temperature. The cell growth is reversibly arrested at the G1/S boundary before entering S phase. Thus, the Cdc7 function is essential for DNA replication in budding yeast. A bypass mutation (bob1) of cdc7 or dbf4 function was reported (Hardy et al. 1997). bob1 was mapped in mcm5 (P83L), supporting the importance of phosphorylation of MCM in Cdc7-mediated regulation of initiation. The mutation may cause conformational changes in the MCM complex which may be permissive for origin activation in the absence of Cdc7 (Hoang et al. 2007). More recently, deletion of an N-terminal segment of MCM4 was shown to bypass Cdc7 function (Sheu and Stillman 2010). It was proposed that the sole essential function of Cdc7 in budding yeast is to relieve an inhibitory activity residing within this N-terminal domain of MCM4 (Sheu and Stillman 2010).
In contrast, the situation may be different in other species. In fission yeast, hsk1 (Cdc7 counterpart in fission yeast) null cells can enter S phase, but eventually replication ceases (Masai et al. 1995). It was a surprise when Δhsk1 was able to grow, albeit at a reduced rate, when combined with Δmrc1 or Δcds1, mutants that are defective in replication stress checkpoint (Hayano et al. 2011). It was then discovered that Δhsk1, which does not form colonies at temperature below 30°C, was able to grow at 37°C. Under these conditions, late/dormant origins are fired, reflecting increased replication potential (Matsumoto et al. 2011). Search for genetic mutation that can bypass Hsk1 function for growth led to identification of rif1, a conserved telomere-binding factor (Hayano et al. 2012). In Δrif1 cells, late/dormant origins were vigorously activated. Δrif1 efficiently restored the growth of Δhsk1. These results indicate that cells can replicate their genome in the absence of Hsk1 under certain genetic or environmental conditions. Δrif1 can partially suppress a cdc7ts in budding yeast as well (Peace et al. 2014; Hiraga et al. 2014).
In mice, Cdc7 knockout results in early embryonic death (E3.5~6.5), probably reflecting its strict requirement for development of inner cell mass. In fact, in vitro culture of Cdc7 knockout of blastocysts failed to generate ICM (inner cell mass). This is due to both inefficient cell growth and to active p53-dependent cell death induced by defective DNA replication. Indeed, Cdc7 p53 double knockout embryo exhibited extended embryonic growth (up to 9 days) and significant ICM formation in vitro (Kim et al. 2002). On the cell level, Cdc7 is essential for growth of mice embryonic stem cells. In mouse embryonic fibroblast cells, Cdc7 knockout reduced DNA replication, and cells underwent senescence (Kim et al. 2003). It remains to be seen whether Cdc7 is essential for growth of all the cell types in mammals. Cdc7 is dispensable in fission yeast cells and possibly in budding yeast cells under certain genetic or environmental conditions. Cells have a way to replicate their genome in the absence of Cdc7. Under the condition Cdc7 is not present, some other kinase may be replacing it.
Cdc7 as a Target for Novel Cancer Therapy
Inhibition of Cdc7 expression in normal somatic cells generally causes stable G1 arrest (Tudzarova et al. 2010; Montagnoli et al. 2004). On the other hand, inhibition of Cdc7 in cancer cell lines causes robust cell death regardless of the p53 status (Ito et al. 2008; Sawa and Masai 2009; Rodriguez-Acebes et al. 2010). Generally in p53-negative background, cells tend to get arrested at G2 and then proceed into aberrant mitosis, whereas p53-positive cancer cells have tendency to proceed into aberrant S phase, before they die (Ito et al. 2012). These incidents happen because of deficiency in various checkpoint systems found in cancer cells. Efforts are being made to develop specific chemical inhibitors of Cdc7 kinase and use them as potent agents that may induce cancer cell-specific cell death (Ito et al. 2008; Sawa and Masai 2009; Montagnoli et al. 2010; Swords et al. 2010; Irie et al. 2017). Indeed, panels of low molecular weight chemical compounds have been identified that inhibit Cdc7 kinase in vitro and can suppress tumor growth in xenograft cancer model mice (Montagnoli et al. 2008). Those who are interested in learning more about eukaryotic DNA replication are recommended to read the reference (Masai et al. 2010).
Conclusions and Future Perspectives
Cdc7 is a serine-threonine kinase conserved from yeasts to human and regulates initiation of DNA replication by phosphorylating MCM, essential components of pre-replicative complexes generated at prospective replication origins. This phosphorylation triggers recruitment of Cdc45 and other replisome proteins to generate active replication forks. Cdc7 forms a complex with an activation subunit Dbf4/ASK to generate an active kinase. In addition to DNA replication, Cdc7 regulates varieties of chromosome dynamics including meiotic DNA recombination, meiotic cell division, DNA damage repair through trans-lesion DNA synthesis, DNA replication checkpoint, and chromatin structures. Cdc7 emerges as a novel cancer therapy target since inhibition of Cdc7 in cancer cells causes efficient cell death by inducing abortive DNA synthesis or aberrant mitosis while that in normal cells generally arrests the cell growth in G1.
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Masai, H. (2018). Cdc7. In: Choi, S. (eds) Encyclopedia of Signaling Molecules. Springer, Cham. https://doi.org/10.1007/978-3-319-67199-4_593
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