Hsk1 (homologue of Cdc7 kinase 1) of the fission yeast is a member of the conserved Cdc7 (cell division cycle 7) kinase family, and promotes initiation of chromosome replication by phosphorylating Mcm (minichromosome maintenance) subunits, essential components for the replicative helicase. Recent studies, however, indicate more diverse roles for Hsk1/Cdc7 in regulation of various chromosome dynamics, including initiation of meiotic recombination, meiotic chromosome segregation, DNA repair, replication checkpoints, centromeric heterochromatin formation and so forth. Hsk1/Cdc7, with its unique target specificity, can now be regarded as an important modulator of various chromosome transactions.

Introduction

Cdc7 (cell division cycle 7) was originally identified in the budding yeast Saccharomyces cerevisiae as one of cdc mutants of the Hartwell collection [1]. Its terminal phenotype as well as the fact that protein synthesis is no longer required for completion of S-phase after the execution of Cdc7 function led to the notion that Cdc7 would function immediately before the onset of S-phase [1,2]. Cdc7 was later identified as a serine/threonine kinase [3] and found to form a complex with the Dbf4 subunit [4]. The orthologue of Cdc7 in other species had not been known until Hsk1 (homologue of Cdc7 kinase 1) was identified in the fission yeast Schizosaccharomyces pombe, which was followed by discovery of related kinases from higher eukaryotes including humans. Dbf4 subunit homologues have also been shown to be conserved across species [Dfp1 (Dbf4 in S. pombe)/Him1 (Hsk1-interacting molecule) in fission yeast; see Table 1 for a comparison of the names of various homologues]. Cdc7 complexes are now known to be widely conserved in evolution and to play crucial roles in initiation of DNA replication.

Table 1
Names of the orthologues in the three species that appear in the present review
Budding yeast Fission yeast Mammal 
Cdc7 Hsk1 Cdc7 
Dbf4 Dfp1/Him1 ASK/Dbf4 
 Swi6 HP1 
Mer2 Rec15  
Spo11 Rec12 Spo11 
Rec114 Rec7 Rec114 
Mei4 Rec24 Mei4 
Lrs4 Mde4  
Mrc1 Mrc1 Claspin 
Mec1 Rad3 ATR 
Rad53 Cds1 Chk1 
Tof1 Swi1 Timeless/Tim1 
Csm3 Swi3 Tipin 
Budding yeast Fission yeast Mammal 
Cdc7 Hsk1 Cdc7 
Dbf4 Dfp1/Him1 ASK/Dbf4 
 Swi6 HP1 
Mer2 Rec15  
Spo11 Rec12 Spo11 
Rec114 Rec7 Rec114 
Mei4 Rec24 Mei4 
Lrs4 Mde4  
Mrc1 Mrc1 Claspin 
Mec1 Rad3 ATR 
Rad53 Cds1 Chk1 
Tof1 Swi1 Timeless/Tim1 
Csm3 Swi3 Tipin 

An essential role for Cdc7 in initiation of DNA replication is to phosphorylate components of pre-RC (pre-replicative complex), which is assembled in the previous G1-phase. Pre-RC is generated by loading of the Mcm (minichromosome maintenance) complex on to the ORC (origin recognition complex) which binds at the potential replication origins in a manner dependent on Cdc6 and Cdt1. Mcms are common substrates of Cdc7, and this phosphorylation as well as that mediated by Cdk (cyclin-dependent kinase) facilitates the assembly of helicase-active CMG (Cdc45–Mcm–GINS) complexes at the origins [5].

In addition to this well-established conserved role of Cdc7 in initiation of DNA replication, it has been known that Cdc7 also plays important roles in various chromosome transactions, including initiation of meiotic recombination, bypass of DNA synthesis, sister chromatid cohesion, centromeric heterochromatin formation, Caf1-mediated chromatin formation and replication checkpoint regulation. In the present short review, we take fission yeast Hsk1 as an example to overview its versatile roles that are important for fine regulation of various chromosome dynamics.

Identification of Hsk1–Dfp1/Him1

Both CDC7 and DBF4 were originally identified in budding yeast as cell cycle mutants that arrest immediately before S-phase [1,2,68]. The fission yeast homologue of Cdc7, named hsk1, was identified by cross-species hybridization and degenerate oligonucleotide-directed PCRs [9] (Figure 1A). A Dbf4 homologue was also first reported in fission yeast as a protein co-purified with Hsk1 [10] and named Dfp1. It was also independently identified through a two-hybrid screening for Hsk1-interacting factor and named Him1 [11]. Expression of Dfp1/Him1 oscillates during the cell cycle, peaking at the G1/S boundary, similar to the budding yeast DBF4, the transcript of which shows periodic regulation within the cell cycle [12,13]. The Hsk1 protein level may also slightly oscillate during the cell cycle.

Schematic drawing and comparison of Cdc7 and Dbf4 subunits from fission yeast, budding yeast and humans

Figure 1
Schematic drawing and comparison of Cdc7 and Dbf4 subunits from fission yeast, budding yeast and humans

(A) Comparison of Cdc7 orthologues. Red and purple segments represent the conserved kinase domains [72] and less conserved kinase insert sequences respectively. (B) Comparison of Dbf4 orthologues. Purple, green and orange segments represent conserved Dbf4-motifs, N, M and C respectively [17]. Sequences fitting to an HP1-binding motif (PXVXL/I/V) are shown with thick vertical bars. Only the one in S. pombe has been shown to be of functional significance, and others have not been evaluated.

Figure 1
Schematic drawing and comparison of Cdc7 and Dbf4 subunits from fission yeast, budding yeast and humans

(A) Comparison of Cdc7 orthologues. Red and purple segments represent the conserved kinase domains [72] and less conserved kinase insert sequences respectively. (B) Comparison of Dbf4 orthologues. Purple, green and orange segments represent conserved Dbf4-motifs, N, M and C respectively [17]. Sequences fitting to an HP1-binding motif (PXVXL/I/V) are shown with thick vertical bars. Only the one in S. pombe has been shown to be of functional significance, and others have not been evaluated.

Fission yeast is unique in that it has another Cdc7 kinase complex, the meiosis-specific Spo4–Spo6 complex. Both spo4+ and spo6+ are expressed specifically during meiosis and are non-essential for mitotic cell growth [14].

Regulation of Hsk1 activity by Dfp1/Him1

Unlike Cdc7 from budding yeast or human, Hsk1 has a basal kinase activity on its own. Dfp1/Him1 stimulates the kinase activity and also may facilitate substrate recognition [15,16]. Although the protein level of Hsk1 is rather constant throughout the cell cycle (it slightly increases during S-phase), the abundance of Dfp1/Him1 protein and its transcript varies during progression through the cell cycle. The Dfp1/Him1 protein, absent from G1-phase, appears at the G1/S transition, coincident with the initiation of DNA replication, and the protein level is maintained throughout the S-phase [11,15].

Comparison of the amino acid sequences of Dbf4 homologues from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4 motifs-N, -M and -C [16,17] (Figure 1B). Dbf4 motif-M, a unique proline-rich motif, and Dbf4 motif-C, a C2H2-type zinc-finger motif, are essential for mitotic functions of Dfp1/Him1 protein as well as for full-level activation of Hsk1 (motif-M alone can support viability in a plasmid loss assay, but motif-C is required for viability through meiosis). In vitro, a small segment containing the Dbf4 motif-M or -C alone binds to and partially activates Hsk1. Co-expression of these two segments augments the extent of activation. Furthermore, a fused polypeptide containing only Dbf4 motifs-M and -C without any spacer can activate Hsk1 and can rescue the growth defect of dfp1/him1-null cells. Deletion of Dbf4 motif-N, which has some similarity with the BRCT [BRCA (breast cancer early-onset 1) C-terminal] domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methanesulfonate, whereas mitotic functions and kinase activation are intact [11,16]. It was suggested that the budding yeast Dbf4 has a role in targeting Cdc7 to replication origins [18]. Dbf4 Motif-N of Dfp1/Him1 is involved in interaction with replication origins in vivo [16].

In budding yeast, it was shown that the N-terminal 320 residues of Dbf4 are required for an in vivo one-hybrid interaction with origins [18] and that the N-terminal 296 residues of Dbf4 interact with Orc2 and Orc3 subunits of the ORC [19], suggesting the involvement of motif-N in ORC interaction. However, another study shows that a Dbf4 mutant lacking the N-terminal 221-residue region spanning motif-N can interact with the ORC as efficiently as the full-length protein [20].

Regulation of DNA replication by Hsk1/Cdc7

Phosphorylation of Mcm2, Mcm4 and Mcm6 by Cdc7 in combination with Cdk and a checkpoint kinase promotes recruitment of the initiation factor Cdc45 to licensed origins [2123] (Figure 2). Thus Cdc7-dependent Cdc45 recruitment is crucial for establishing replication forks during S-phase [2228]. Hsk1 phosphorylates N-terminal tail regions of Mcm2, Mcm4 and Mcm6. Phosphorylated forms of Mcm4 and Mcm6 were selectively co-immunoprecipitated with Cdc45, suggesting that phosphorylation triggers binding of Cdc45. Combination of Mcm4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of Mcm2 and Mcm6 respectively, containing clusters of serine/threonine, led to an apparent non-viable phenotype, suggesting that the phosphorylation of the tail regions of different Mcm subunits may be redundant in their function for Cdc45 recruitment [22].

Hsk1/Cdc7 is a modulator of many chromosome transactions

Figure 2
Hsk1/Cdc7 is a modulator of many chromosome transactions

Various chromosome dynamics that are regulated by Cdc7 are shown. Roles for Cdc7 in histone modification and chromatin reconstitution have been studied only in mammalian cells. Regulation of Rad9 has been reported only in fission yeast. The events of phosphorylation mediated by Cdc7 that have been identified in vivo or in vitro are indicated by circled Ps. Grey arrows indicate the proteins recruitment upon phosphorylation by Hsk1/Cdc7. For meiotic recombination, recruitment of Spo11 is speculated.

Figure 2
Hsk1/Cdc7 is a modulator of many chromosome transactions

Various chromosome dynamics that are regulated by Cdc7 are shown. Roles for Cdc7 in histone modification and chromatin reconstitution have been studied only in mammalian cells. Regulation of Rad9 has been reported only in fission yeast. The events of phosphorylation mediated by Cdc7 that have been identified in vivo or in vitro are indicated by circled Ps. Grey arrows indicate the proteins recruitment upon phosphorylation by Hsk1/Cdc7. For meiotic recombination, recruitment of Spo11 is speculated.

The kinase Hsk1–Dfp1/Him1 has been shown to be a rate-limiting activator of origins. The level of Hsk1–Dfp1/Him1 correlated with origin firing efficiency [29,30]. Furthermore, tethering Hsk1–Dfp1/Him1 near an origin increases the efficiency of the tethered origin [29]. These data suggest that the accessibility of replication origins to Hsk1–Dfp1/Him1 may regulate origin efficiency.

Heterochromatin is a structurally compact region of chromosomes in which transcription and recombination are inactivated. It is also involved in temporal regulation of DNA replication. Among heterochromatin loci in fission yeast, the pericentromeric region and the silent mating-type (mat) locus replicate in early S-phase, whereas the sub-telomeric region replicates in late S-phase [31]. Swi6, a S. pombe counterpart of HP1 (heterochromatin protein 1), is required for early replication of the pericentromeric region and the mat locus. Hsk1–Dfp1/Him1 interacts with Swi6 through P448VVTI452 of Dfp1/Him1, an HP1-binding motif (PXVXL/I/V) [31,32] and this HP1-binding motif is required for early replication of the pericentromeric region and mat locus. Tethering Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci [31]. These observations also reinforce the idea that Hsk1/Cdc7 is a critical determinant of replication timing.

Regulation of meiotic recombination by Hsk1/Cdc7

In an hsk1 temperature-sensitive strain (hsk1-89), meiosis is arrested with one nucleus state before meiosis I in most of the cells and meiotic recombination frequency is reduced by one order of magnitude, whereas pre-meiotic DNA replication is delayed but is apparently completed. Strikingly, formation of meiotic DSBs (double-strand DNA breaks) is largely impaired in hsk1-89, and Hsk1 activity is essential for these processes. Deletion of all three checkpoint kinases, i.e. Cds1, Chk1 and Mek1, does not restore DSB formation, meiosis or Cdc2 activation, which is suppressed in hsk1-89, suggesting that these aberrations are not caused by known checkpoint pathways, but that Hsk1 itself appears to regulate DSB formation and meiosis. Whereas transcriptional induction of some rec genes and horsetail movement are normal, chromatin remodelling at ade6-M26, a recombination hotspot, which is a prerequisite for subsequent DSB formation at this locus, is not observed in hsk1-89. These data indicate essential roles for Hsk1 in the initiation of meiotic recombination and meiosis [33].

A dfp1 mutant, dfp1(1-519), which lacks the C-terminal zinc finger, causes severe meiotic defects, including reduced spore viability, reduced formation of programmed DSBs, altered expression of meiotic genes and disrupted chromosome segregation. There is a high frequency of dyad formation [34]. A similar naturally occurring C-terminal truncation mutant of dfp1 (rad35) was previously identified and was shown to be sensitive to DNA damages.

Potential roles for Cdc7 in meiosis had been suggested more than 30 years ago in budding yeast [35]. It was reported that Cdc7 may be required for synaptonemal complex formation during meiotic recombination (Figure 3). More recent analyses showed that Cdc7 in budding yeast is essential for meiotic DSBs and meiosis I progression [36,37]. The DNA topoisomerase-II-like endonuclease Spo11 catalyses the formation of DSBs in DNA. The phosphorylation by Cdc7 on Ser29 of Mer2, a Spo11 accessory protein, is essential for meiotic DSB formation and is primed by the phosphorylation of Mer2 Ser30 by S-CDK (S-phase CDK). Diploids of cdc7Δ display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic co-activators that may assist Spo11 binding to DSB hotspots [38,39]. Thus phosphorylation of Mer2 by Cdc7 is essential for recruitment of the Spo11 endonuclease complex to the provisional DSB sites. Pontential targets of Hsk1 during initiation of meiotic recombination in fission yeast are still unknown.

Roles of Cdc7 during meiosis

Figure 3
Roles of Cdc7 during meiosis

The kinase Cdc7 has been shown to play crucial roles during meiosis in yeasts and mammals. This Figure summarizes studies in budding and fission yeasts. Unlike the mitotic cell cycle, Cdc7 does not play essential roles during pre-meiotic DNA replication. However, it plays crucial roles in initiation of meiotic recombination; namely the formation of DSBs. Furthermore, it has been shown that Cdc7 also plays essential roles in meiosis I progression. Information from budding yeast is in green, that from fission yeast in blue, and what has been shown to be true for both yeasts is in red. Fission yeast has another Cdc7-related kinase complex, Spo4–Spo6, which only functions during meiosis II and sporulation. Its substrates are not known.

Figure 3
Roles of Cdc7 during meiosis

The kinase Cdc7 has been shown to play crucial roles during meiosis in yeasts and mammals. This Figure summarizes studies in budding and fission yeasts. Unlike the mitotic cell cycle, Cdc7 does not play essential roles during pre-meiotic DNA replication. However, it plays crucial roles in initiation of meiotic recombination; namely the formation of DSBs. Furthermore, it has been shown that Cdc7 also plays essential roles in meiosis I progression. Information from budding yeast is in green, that from fission yeast in blue, and what has been shown to be true for both yeasts is in red. Fission yeast has another Cdc7-related kinase complex, Spo4–Spo6, which only functions during meiosis II and sporulation. Its substrates are not known.

Regulation of meiotic cell division by Hsk1/Cdc7 and related kinases

During meiosis I, sister kinetochores attach to microtubules from the same spindle pole body (mono-orientation), rather than to those from opposite spindle pole bodies (biorientation). In budding yeast, Cdc7 is essential for the localization of the monopolin complex to kinetochores, which is required for mono-orientation, probably through phosphorylation of the monopolin subunit Lrs4 [40,41]. In fission yeast, the localization of the monopolin complex to kinetochores is not required for mono-orientation [42]. Rec8 phosphorylation by both CK1 (casein kinase 1) and Hsk1/Cdc7 is a prerequisite for the cleavage of Rec8 by separase in budding and fission yeasts [34,43,44].

The meiosis-specific Cdc7-like kinase complex in fission yeast, Spo4–Spo6, is dispensable for mitotic growth and premeiotic DNA replication. spo4-null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented in these mutants [14]. In contrast, another recent report showed that abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus in the absence of Spo4 or Spo6 [45]. Thus Spo4–Spo6 may have a critical function in regulating the spindle length during meiosis II.

Regulation of DNA repair by Hsk1/Cdc7

It has long been known in budding yeast that cdc7 mutants show various degrees of UV sensitivity and UV-induced mutagenesis [4648]. Fission yeast mutants of hsk1 and dfp1 are also defective for MMS (methyl methanesulfonate)-induced mutagenesis [49]. Conversely, yeast strains engineered to express high levels of Cdc7 show hypermutability in response to UV [50]. Genetic interactions between Cdc7 and members of the TLS (translesion synthesis) pathway have been described in budding yeast [51]. Thus Cdc7 may directly regulate the TLS pathway, although the potential targets have not been elucidated in yeasts.

The roles for Cdc7 in TLS of human cells have recently been described. The E3 ubiquitin ligase Rad18 guides DNA Polη (polymerase η) to sites of replication fork stalling and monoubiquitinates PCNA (proliferating-cell nuclear antigen) to facilitate binding of Y family TLS DNA polymerases during TLS in human cells. A serine cluster in the Polη-binding motif of Rad18 is phosphorylated by Cdc7 in human cells. Efficient association of Rad18 with Polη is dependent on Cdc7, and Cdc7 facilitates redistribution of Polη to sites of replication fork stalling [52].

In fission yeast, Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1 in response to replication-induced DNA damage. Phosphorylation of Rad9 by Hsk1 depends on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5 and prior phosphorylation by Rad3, and disrupts its interaction with RPA (replication protein A). rad9 mutants defective in Hsk1 phosphorylation show wild-type checkpoint responses, but abnormal DNA repair protein foci and decreased viability after replication stress. Rad9 phosphorylation by Hsk1 probably releases Rad9 from DNA damage sites to facilitate DNA repair [53].

Hsk1 and Dfp1 are associated with the chromatin even after S-phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS-sensitive mutants identified a truncation allele, dfp1(1-519). Hsk1–Dfp1 functions in replication checkpoint with the Swi1–Swi3 complex [also known as FPC (fork protection complex)] [54,55]. On the other hand, MMS-induced mutagenesis occurs in swi1 or swi3 mutants, but not in hsk1 or dfp1 mutants, suggesting that Hsk1–Dfp1 acts independently of the Swi1–Swi3 complex to promote DNA repair [49].

Regulation of chromosome partition by Hsk1/Cdc7

hsk1 mutants often show highly disordered nuclear morphology at their restriction temperature [54,5658]. The cdc25-22 single mutant can help the cell cycle proceed when released at 30°C. The hsk1-89 cdc25-22 double mutant arrested in late G2-phase can complete mitosis when released at 25°C, but cannot proceed through mitosis, with abnormally segregated chromosomes being accumulated when released at 30°C, a non-permissive temperature of hsk1-89 (S. Matsumoto, unpublished work), suggesting a critical role for Hsk1 in mitotic chromosome segregation.

rad21+ encodes a non-SMC subunit (Kleisin) of the mitotic cohesin complex. A rad21-K1 hsk1 double mutant exhibits a severe synthetic growth defect [56,57]. hsk1-89 exhibits the premature separation of sister chromatids [56] and dfp1(1-519), a dfp1 C-terminally truncated mutant, is defective in the phosphorylation and degradation of Rec8, the meiotic counterpart of Rad21, resulting in a failure to proceed through the MII division [34]. These results are consistent with a crucial role for hsk1+-dfp1+/him1+ during M-phase through interaction with cohesin.

In budding yeast, a short N-terminal Dbf4p segment targets Cdc7p–Dbf4p to the Polo kinase Cdc5p. Dbf4p interacts with the Polo substrate-binding domain and inhibits its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the MEN (mitotic exit network), presumably by altering Polo substrate targeting. Cdc7p–Dbf4p may prevent inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint [59]. In fission yeast, hsk1-89 plo1ts shows synthetic growth defect, possibly suggesting functional interactions between the Polo kinases and Hsk1 (S. Matsumoto and H. Masai, unpublished work). Cdc7 interacts with Polo kinases in human cells as well and regulates the cellular localization of the latter kinase (S. Ito, G.-T. Toh and H. Masai, unpublished work).

Regulation of protein degradation by Hsk1/Cdc7

Mrc1 is one of the targets of Hsk1 and it was shown that it is stabilized during S-phase in the hsk1-89 mutant. Further analyses identified a phospho-degron on Mrc1 (residues 859–864), which is phosphorylated by Hsk1 and triggers proteasome-dependent degradation of Mrc1 [60].

A similar Hsk1-mediated protein degradation was reported. Hsk1 phosphorylates Ams2, a cell-cycle-regulated GATA-type transcription factor. SCF(Pof3)-mediated degradation of Ams2 is responsible for cell-cycle-dependent transcriptional activation of all of the core histone genes peaking at G1/S-phase and prevents abnormal incorporation of canonical H3 into the central CENP-A (centromere protein A)/Cnp1-rich centromere [61]. Hsk1 plays a vital role during post-S-phase in maintaining centromere integrity and genome stability through inducing degradation of Ams2 by site-specific phosphorylation [61].

Regulation of replication checkpoint by Hsk1/Cdc7

In response to DNA damage or replication fork stalling, the intra-S-phase checkpoint inhibits the firing of late replicating origins as well as replication fork rate. In budding yeast, the replication factor Sld3 is phosphorylated by Rad53, and this phosphorylation, along with phosphorylation of Dbf4 by Rad53, blocks late origin firing. Upon exposure to DNA-damaging agents, cells expressing non-phosphorylatable Sld3 and Dbf4 proceed through S-phase faster than wild-type cells by inappropriately firing late origins, grow poorly showing multiple Rad52 foci in the presence of hydroxyurea, and exhibit delayed recovery from the replication block and subsequently arrest at the DNA damage checkpoint [62,63].

In fission yeast, Dfp1/Him1 is phosphorylated in vivo upon incubation with hydroxyurea, and this phosphorylation depends on active Cds1 [11,15], suggesting that Dfp1/Him1, as is the case for Dbf4 in budding yeast, is a target of Cds1-mediated response to replication arrest. Hsk1 also undergoes Cds1-dependent phosphorylation in response to hydroxyurea and can be phosphorylated by purified Cds1 kinase in vitro, suggesting that the Hsk1 could also be a potential target of Cds1 regulation [57].

On the other hand, Hsk1–Dfp1/Him1 appears to have a critical role in regulating checkpoint. Hsk1 is also required for Cds1 activation [56]. Hsk1 genetically and physically interacts with Swi1–Swi3 and Mrc1, mediators of Cds1 activation and also components of the replication fork protection complex [54,60,64]. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Hsk1 vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ (serine-glutamine/threonine-glutamine) sites. The replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in hsk1-89 and in goa1, an initiation-defective mutant of cdc45, but not in a mcm2 or polϵ mutant, suggesting that Hsk1-mediated loading of Cdc45 on to replication origins may play important roles in replication-stress-induced checkpoints [65] as Cdc7 does in budding yeast [63].

Bypass of Hsk1 revealed the plasticity in origin firing program in fission yeast

In budding yeast, bob1, an allele of mcm5, can bypass the requirement of cdc7 and dbf4 for DNA replication and growth [66]. More recently, deletion within the N-terminal segment of Mcm4 was shown to bypass cdc7 in budding yeast [28].

In fission yeast, no mcm mutants have been isolated so far which can suppress hsk1 or dfp1/him1 mutants. In contrast, a growth defect of hsk1-89 at a non-permissive temperature is partially suppressed by cds1Δ [54,57]. Recently, it was discovered that mrc1Δ not only suppresses the temperature-sensitive growth of hsk1-89, but also restores the viability of hsk1Δ at 30°C. Furthermore, cds1Δ or a checkpoint-deficient mutant (mrc1-3A) of mrc1 was found to suppress hsk1Δ, albeit to a lesser extent compared with mrc1Δ [58]. In mrc1Δ cells, some late origins are fired in the presence of hydroxyurea. Out of approximately 600 late/dormant origins, approximately 100 origins are fired in the presence of hydroxyurea in mrc1Δ cells. An almost identical set of origins are activated in cds1Δ cells as well. Interestingly, in mrc1Δ, firing efficiency and timing is advanced selectively at early-firing origins that are normally bound by Mrc1 [67]. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in cds1Δ or mrc1-3A, suggesting that non-checkpoint function of Mrc1 is involved in maintaining the normal programme of early-firing origins [67]. More recently, we identified rif1Δ as an efficient bypass mutant of hsk1Δ [68]. In rif1Δ, extensive deregulation of dormant origins over a wide range of chromosomes occurs in the presence or absence of hydroxyurea. At the same time, many early-firing efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that do not overlap with the pre-RC assembly sites, although many of the Rif1-binding sites are located in the vicinity of the late/dormant origins suppressed by Rif1. Whereas Rif1 binding to telomeres depends on Taz1, that to the arm segments is independent of Taz1 [68], suggesting that the mode of Rif1 binding may be distinct in the arm segments. Regulation of the replication timing by Rif1 is also conserved in mammalian cells. Rif1 appears to regulate the structures of replication timing domains through its ability to organize chromatin loop structures in mammalian cells [69,70]. Rif1 was originally identified in budding yeast as a telomere-binding protein. Another telomere-binding protein, Taz1, was also reported to control replication timing through its localization near late replication origins in fission yeast [71]. In contrast with rif1Δ, taz1Δ cannot bypass the growth defect of hsk1 mutants [68].

Unexpectedly, we found that hsk1Δ is viable at 37°C. More DNA is synthesized and some dormant origins fire in the presence of hydroxyurea at 37°C compared with at a lower temperature. Furthermore, the hsk1Δ bypass strains hsk1Δ mrc1Δ and hsk1Δ cds1Δ grow poorly at 25°C compared with at higher temperatures [58]. Although the mechanisms are not clear, high temperature facilitates initiation of fission yeast chromosome replication. These observations provided evidence for plasticity of the replication programme in fission yeast that can change under different physiological and genetic conditions.

Summary and perspectives

Nearly 45 years after the initial discovery of cdc7 by Hartwell [1], the functions of the kinase Cdc7 now go far beyond simply regulating replication initiation. It appears to be involved in almost all aspects of chromosome dynamics and maintenance of chromosome integrity during cell cycle. Studies of the Hsk1–Dfp1/Him1 complex in fission yeast have greatly contributed to the understanding of novel functions of this conserved kinase. Lack of a consensus target sequence of Cdc7 other than its general preference for an acidic environment surrounding the target serine/threonine residues [21] has hampered the informatics-based search of potential Cdc7 target molecules. However, now many new substrates for Cdc7/Hsk1 have been identified, a consensus view on how Cdc7 works has started to emerge. It generally phosphorylates multiple residues in the target segments of the substrate protein and this may cause the change in protein conformation or charge distribution on the protein surface, which may attract other proteins to further modify the chromatin–DNA complexes for execution of various chromosome dynamics.

One unexpected finding from the studies on fission yeast Hsk1 is that cells can initiate DNA replication without Hsk1 under variety of conditions. In that sense, Hsk1 is not essential for the firing of origins, but just increases the probability of origin firing by phosphorylating Mcm subunits. If the probability of origin firing is increased by other means, the requirement for Cdc7 may be lowered, permitting the bypass of this kinase for initiation. Thus Cdc7 can be regarded as a ‘modulation’ kinase that phosphorylates varieties of target proteins containing a rather promiscuous acidic surface and facilitates the decision-making process for chromosome fate. This sort of regulation by Cdc7 may also be fitted for stochastic regulation of chromosome dynamics.

The 7th International Fission Yeast Meeting: Pombe 2013: An Independent Meeting/EMBO Conference held at University College London, London, U.K., 24–29 June 2013. Organized and Edited by Jürg Bähler (University College London, U.K.) and Jacqueline Hayles (Cancer Research UK London Research Institute, U.K.).

Abbreviations

     
  • Cdc

    cell division cycle

  •  
  • Cdk

    cyclin-dependent kinase

  •  
  • DSB

    double-strand DNA break

  •  
  • Him1

    Hsk1-interacting molecule

  •  
  • HP1

    heterochromatin protein 1

  •  
  • Hsk1

    homologue of Cdc7 kinase

  •  
  • Mcm

    minichromosome maintenance

  •  
  • MMS

    methyl methanesulfonate

  •  
  • ORC

    origin recognition complex

  •  
  • Polη

    polymerase η

  •  
  • pre-RC

    pre-replicative complex

  •  
  • TLS

    translesion synthesis

We apologize to those authors whose important papers could not be cited in this article due to limitations of space. We especially apologize that we were not able to cite many papers related to Cdc7 functions in organisms other than fission yeast. We thank Yutaka Kanoh, Motoshi Hayano, Michie Shimmoto, Naoko Kakusho, Rino Fukatsu and Kyosuke Ueda for contribution to our studies described in the present review, and other members of our laboratory for helpful discussion.

Funding

This work was supported in part by Grants-in-Aid for Basic Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant number 23247031 (to H.M.)].

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