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Genes to Cells (2005) 10, 551-563. doi:10.1111/j.1365-2443.2005.00857.x
© 2005 Blackwell Publishing or its licensors
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Functional analyses of mouse ASK, an activation subunit for Cdc7 kinase, using conditional ASK knockout ES cells

Nobuyuki Yamashita1,2, Jung-Min Kim1, Osamu Koiwai2, Ken-ichi Arai3,4 and Hisao Masai1,*

1 Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan
2 Department of Applied Biological Science, Tokyo University of Science, Chiba 278-8510, Japan
3 Department of Integral Biological Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan
4 Gingko Biomedical Research Institute, Kawasaki, Kanagawa 216-0001, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
ASK (activator of S phase kinase) is an activation subunit for mammalian Cdc7 kinase. We have generated mutant ES cell lines in which ASK can be conditionally inactivated. Upon loss of the ASK genes, the mutant ES cells rapidly cease cell growth. In keeping with its expected roles in activation of the essential S phase kinase, DNA synthesis is arrested and significant cell death is eventually induced in ASK-deficient cells, demonstrating essential roles of ASK for viability of ES cells. Using these mutant cells, we have set up a system where ASK molecules can be functionally dissected. In keeping with previous results from yeasts, conserved motif-M and motif-C were shown to be essential for in vivo functions of ASK, whereas a long C-terminal tail, found only in ASK-related molecules in higher eukaryotes, is not required. Unexpectedly, the motif-N, related to the BRCT motif and dispensable for viability in yeasts, is essential for the viability of ES cells. Further characterization reveals that motif-N is required for the maximum phosphorylation of MCM in cells, whereas the autophosphorylation activity of Cdc7 is not significantly affected by its loss. These results may suggest that motif-N of ASK may facilitate recruitment of substrates for Cdc7 kinase.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
ASK (activator of S phase kinase) is a mammalian homolog of Dbf4 of budding yeast (Dfp1/Him1 in fission yeast) (Yoon et al. 1993; Brown & Kelly 1998; Oshiro et al. 1999; Takeda et al. 1999), and is an activation subunit of Cdc7 kinase (Hsk1 in fission yeast). Cdc7, a serine/threonine kinase, is required for S phase initiation in yeasts (Sclafani & Jackson 1994; Sclafani 2000; Masai & Arai 2002). Genetic and biochemical evidence indicates that one of the significant substrates of Cdc7 may be the MCM complex. Among the six MCM subunits, MCM2 is most efficiently phosphorylated by Cdc7 in vitro (Lei et al. 1997; Kumagai et al. 1999; Kihara et al. 2000; Masai et al. 2000; Sclafani 2000). Essential roles of Cdc7 in mammalian cell proliferation were demonstrated by generation of conditional Cdc7 knockout cell lines. Upon loss of functional Cdc7 protein, the mutant cells ceased DNA synthesis, followed by p53-dependent cell death (Kim et al. 2002).

Recent studies indicate the presence of multiple Dbf4-related molecules with distinct functions, suggesting the presence of novel ‘Dbf4-family’ proteins (Johnston et al. 1999; Masai & Arai 2000; Montagnoli et al. 2002; Nakamura et al. 2002; Furukohri et al. 2003; Yanow et al. 2003; Yoshizawa-Sugata et al. 2005). Dbf4-related proteins carry three conserved domains named Dbf4-motif-N, -M and -C. Dbf4-motif-N shows some similarity to the domain I of the BRCT (BRCA1 C-terminal) domain, which was shown to be present on wide varieties of repair and DNA damage/replication checkpoint proteins (Bork et al. 1997; Saka et al. 1997; Masai & Arai 2000), and was suggested to interact with chromatin or with origin recognition complex (ORC) (Dowell et al. 1994; Pasero et al. 1999; Jares & Blow 2000) or Rad53 in budding yeast (Duncker et al. 2002). Dbf4-motif-M is a conserved proline-rich domain and is responsible for the interaction with and activation of Cdc7 (Ogino et al. 2001; Fung et al. 2002; Sato et al. 2003). Dbf4-motif-C bears the highest degree of conservation among the three and contains a highly conserved CCHH-type zinc finger motif which is also involved in the interaction with and activation of Cdc7 (Johnston et al. 1999; Masai & Arai 2000). This motif is required also for DNA damage responses during S phsse (Fung et al. 2002). Dbf4-motif-M (and Dbf4-motif-C under some condition) is essential for growth and sufficient for activation of Hsk1 and for proliferation in fission yeast (Ogino et al. 2001; Fung et al. 2002). Similarly, the combination of Dbf4-motif-M and Dbf4-motif-C of human ASK is sufficient for binding with and activation of human Cdc7 in mammalian cells (Sato et al. 2003).

In order to genetically characterize the functions of ASK in mammalian cells, we attempted to generate a mutant mouse ES cell line lacking the genes for mouse ASK (muASK). Toward this goal, we have taken an approach that was successfully employed to generate a conditional muCdc7-deficient ES cell line (Kim et al. 2002) in view of the likelihood that ASK is essential for cell growth. Our approach is to generate complete knockout of the gene of interest in the presence of a ‘removable’ transgene. Our results indicate that muASK is essential for growth of ES cells and its inactivation leads to arrest of DNA synthesis. Using the established muASK mutant cell lines, we were able to functionally assay various deletion and point mutants of muASK protein, which lead to the discovery that Dbf4-motif-N, known to be dispensable for growth in yeasts, is essential for ES cell proliferation.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Strategy for characterization of ASK molecule using a conditional ASK knockout ES cell line

Since cell cycle genes, such as those involved in DNA replication, are likely to be essential for cell proliferation, detailed functional analyses require generation of cell lines in which the gene of interest can be conditionally inactivated, or which contain reduced activity. Our strategy is to generate complete knockout of endogenous genes in the presence of a transgene which is conditionally excisable by the Cre-loxP system.

One allele of the muASK genes was disrupted by insertion of the neomycin resistance gene through homologous recombination in the mouse ES cell line CCE28 (ASK+/– ES cell). Then the Flox transgene vector was introduced into the heterozygous ES cell line (ASK+/–tg1st ES cell). The resulting heterozygous ES cell line expressing the transgene was exposed to the high concentration of G418 to induce gene conversion, leading to generation of ASK–/–tg1st ES cells. The Flox transgene can be removed by the expression of the Cre recombinase, permitting us to examine muASK null phenotypes in mouse ES cells.

Next, a series of truncation, internal deletion and point mutation derivatives of muASK fused to HA tag were constructed and were introduced into the ASK–/–tg1st ES cell as a 2nd transgene. The removal of the 1st transgene by expression of the Cre recombinase will permit the characterization of the phenotypes associated with the generated mutations of ASK (Fig. 1).



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  		Figure 1  The strategy for generation of conditional muASK-deficient ES cell lines carrying a Cre-removable (flox) FLAG-muASK transgene (tg) and characterization of mutant ASKs using the mutant cell line.

 
Conditional disruption of the muASK gene in ES cells

The muASK gene is located on the mouse chromosome 5A1, consisting of 12 exons. In order to elucidate the function of mammalian ASK with the above strategy, we first attempted to disrupt one allele of the muASK genes through homologous recombination. Our strategy was to replace the 9.8 kb genome region, containing the exons 1 and 2 encoding the initiation codon and conserved motif-N region, with a ß-galactosidase-internal ribosome entry site-neomycin resistance (neor) cassette (Fig. 2A). The successful disruption of the muASK locus was confirmed by Southern blotting (Fig. 2B) using the probes specific to the muASK.



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  		Figure 2  Targeted disruption of the muASK gene. (A) Locations of the restriction enzyme sites on the wild-type muASK gene, the targeting vector and the mutant muASK resulting from homologous recombination. The numbered filled boxes indicate exons. (B) Identification of muASK+/– clones by Southern blot analysis. Genomic DNA was digested with BglII or XbaI and hybridized with probe a (5'-probe) or b (3'-probe) indicated by horizontal bars in (A). The hybridizing DNA fragments of wild-type (wt) and mutant (mt) alleles are indicated by arrowheads.

 
The resulting heterozygous ES cells (ASK+/–) grow normally, indicating that loss of one allele can be tolerated in terms of ES cell proliferation. In order to determine whether muASK functions are required for ES cell proliferation, we generated mutant ES cell lines in which inactivation of muASK could be conditionally induced. For this purpose, we constructed a flox muASK transgene, which is flanked by two loxP sites and can be excised with Cre recombinase (Fig. 3A). The cDNA used for the transgene encodes 663 amino acid full length muASK polypeptide tagged at the N-terminus with a FLAG peptide. The transgene vector was transfected into muASK+/– heterozygous ES cells by electroporation, and transformants were selected by puromycin resistance. After 7 day selection with puromycin, transformants growing on the feeder layer were picked and cloned.



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  		Figure 3  Introduction of a flox transgene and gene conversion. (A) A schematic drawing of the structure of muASK flox transgene. (B) Southern blotting analysis of conditional muASK-deficient ES clones. Genomic DNA was digested with BamHI and hybridized with the probe c, indicated in (A). (C) Expression of FLAG-muASKwt transgene and its disappearance by the Ad-Cre infection. ASK+/–tg1st cells infected with Ad-Cre or mock-infected were harvested at 2 days after infection. Expression of Flag-muASKwt was examined by Western analysis using anti-FLAG antibody. The asterisk indicates the band reacting nonspecifically with the antibody. (D) Induction of gene conversion in ASK+/–tg1st cells. Genomic DNA was isolated from the cells indicated and hybridized with the probe a or b shown in Figure 2A. (E) Triton-soluble (S) or -insoluble (P) fractions from the indicated cells were analyzed by immunoblotting with anti-FLAG (upper), anti-muASK (middle) or anti-{alpha}-tubulin (lower).

 
Insertion of the transgene vector was confirmed by the Southern analysis (Fig. 3B) and expression of FLAG-muASKwt was confirmed by immunoblotting with anti-FLAG antibody (Fig. 3C). FLAG-muASKwt was identified as multiple bands on SDS-PAGE due to phosphorylation presumably caused by the associated Cdc7 kinase. Since the flox muASK transgene vector has only one BamHI site, the numbers of the bands hybridizing with probe c indicate those of the inserted transgenes in the chromosomes of the ES cell lines. Most of the puromycin-resistant clones have a single copy of the transgene (Fig. 3B) and we have chosen the clone No. 5 and No. 15 for further analyses. The clone No. 5 and No.15 were either infected with Ad-Cre or mock-infected, and Western analysis with anti-FLAG antibody was conducted. FLAG-muASKwt bands detected in the extracts of mock-infected clones 5 and 15 were not detected in those from Ad-Cre-infected cells (Fig. 3C), indicating that the flox transgene has been excised by the Cre recombinase.

We then converted the endogenous wild-type allele to the mutant allele containing a neomycin marker through gene conversion. This was accomplished simply by elevating the concentration of G418 (Mortensen et al. 1992), which permitted the selection of ES cells homozygous for the targeting allele, since they are expected to display resistance to a higher concentration of G418 due to the presence of two copies of the neor gene. After 18–20 day selection in the medium containing 5 mg/mL G418, surviving colonies were cloned and gene conversion was confirmed by Southern analysis. Three or six clones were obtained from the clone No. 5 or No. 15, respectively. One clone derived from the clone No. 15 underwent the expected gene conversion and both alleles of the endogenous muASK gene were disrupted (ASK–/–tg1st) (Fig. 3D). In contrast, muASK–/– cells could not be established from muASK+/– cells under the same condition (data not shown), suggesting that muASK functions are essential for cell proliferation of ES cells.

muASK is essential for ES cell viability

We subsequently infected these muASK–/–tg1st ES cells with adenoviruses expressing Cre recombinase (Ad-Cre) to remove the flox transgene. Western blotting analyses indicated that FLAG-muASKwt protein level decreased below the detection within a day after infection, in both muASK–/–tg1st and muASK+/–tg1st cells (Fig. 3E). One hundred percent of ES cells were infected by the Ad-Cre and transgene was excised in almost all the cells after infection at moi = 120 (Kim et al. 2002). Following the infection with Ad-Cre, the increase in cell numbers stopped almost immediately (Fig. 4A), whereas muASK+/–tg1st ES cells continued to increase in number under the same condition. However, the growth rate decreased in comparison with non-infected cells, presumably due to the effect of adenovirus infection.



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  		Figure 4  Proliferation, cell cycle profile and DNA synthesis in ASK-deficient cells. (A) The numbers of viable cells were counted at various times (0–4 days) after infection of ASK+/–tg1st or ASK–/–tg1st ES cells with Ad-Cre. (B) Flow cytometry analysis of DNA content of muASK+/–tg1st or muASK–/–tg1st ES cells at 48 h after infection of Ad-Cre. Cre–, before infection of Ad-Cre. (C) ASK–/–tg1st ES cells infected or mock-infected were seeded on 96-well plate (1000 cells per well), and were incubated for 20 min in the presence of BrdU at different times after infection, and its incorporation was measured by ELISA. (D) Phosphorylation states of muMCM2 in ASK-deficient cells. Triton-soluble (S) or -insoluble (P) extracts from the indicated cells were analyzed by immunoblotting with anti-MCM2 antibody. The fast-migrating bands of MCM2 in the Triton-soluble fraction are lost in muASK–/–tg1st cells after infection with Ad-Cre. The lower band which appears in the soluble fraction of ASK–/–tg cells after infection may be generated by Cdk-mediated phosphorylation.

 
We then examined the DNA content of Ad-Cre infected or mock-infected muASK–/–tg1st ES cell line. Fluorescence activated cell-sorter (FACS) analysis of propidium iodide-stained cells indicated that cells with late S to G2/M phase DNA content accumulated in the Ad-Cre infected muASK–/–tg1st cells (Fig. 4B). Incorporation of BrdU in Ad-Cre infected muASK–/–tg1st ES cells was greatly reduced compared to the mock-infected cells (Fig. 4C). We then examined the phosphorylation state of MCM2, a known target of Cdc7 kinase, in the mutant cell line. Cdc7-ASK phosphorylates MCM2 in the MCM2-4-6-7 complex and causes downward mobility shift on SDS-PAGE. In the muASK–/–tg1st ES cells, the lowest band of MCM2, which comigrates with MCM2 phosphorylated by Cdc7 in vitro (data not shown), disappeared at 2 days after infection (Fig. 4D), indicating that the Cdc7-dependent phosphorylation is abrogated in the mutant. These results show that ASK is required for Cdc7 kinase activation in vivo, and indicate that muASK is required for cell proliferation and DNA synthesis in ES cells.

Insertion of 2nd transgene and expression of various mutant ASKs

These results indicate that muASK is essential for mouse ES cell proliferation. In order to examine the roles of domains of ASK protein, we introduced the mutated muASK into the muASK–/–tg1st ES cells. ASK/Dbf4 have three conserved domains named Dbf4-motif-N, -M and -C. Among the ASK and Dfp1/Him1 polypeptides tested, only those carrying both Dbf4-motif-M and -C were able to activate Cdc7 in fission yeast (Ogino et al. 2001; Fung et al. 2002) and in human (Masai & Arai 2000; Sato et al. 2003) in vitro. We constructed a series of truncation, internal deletion and point mutant derivatives of muASK (Fig. 5A). They were transfected into COS-7 cells with or without the plasmid expressing mouse Cdc7, and expression of muASK derivatives were examined by Western blotting. The wild-type muASK generated a series of mobility-shifted bands when co-expressed with Cdc7, representing the autophosphorylation. {Delta}N mutant muASK, lacking motif-N, also generated similar shifted bands when in a complex with the muCdc7. Minimum (containing only motif-M and -C) and NMC (containing motif-N, -M and -C but lacking the C-terminal tail) did not exhibit mobility-shift even in the presence of muCdc7 (Fig. 5B). This is probably due to the loss of the C-terminal region of ASK in minimum and NMC, since the major autophosphorylaton sites on ASK responsible for the mobility-shifts are known to be the clusters of serine and threonine residues at the C-terminal end of ASK protein (Sato et al. 2003). M-mut (DY) muASK is also mobility-shifted, albeit to a lesser extent. M-mut(P) was only slightly mobility-shifted, indicating that the mutated proline residue is important for Cdc7 kinase activation as was previously shown for fission yeast Dfp1/Him1 (Ogino et al. 2001).



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  		Figure 5  Expression of mutant ASKs from 2nd transgene vectors. (A) A schematic drawing of the structure of the 2nd transgene vector and muASK mutant derivatives. HA-tagged mutant muASK derivatives are expressed under the control of the EF1{alpha}-promoter. (B) Expression of HA-tagged muASK mutant polypeptides in COS-7 cells with (+) or without (–) HA-tagged muCdc7. The whole cell extracts from the indicated cells were analyzed by immunoblotting with anti-HA antibody. (C) Expression levels of muASK mutant polypeptides stably expressed in muASK–/–tg1st ES clones. The whole cell extracts from muASK–/–tg1st ES cells expressing the ASK wild-type or mutant polypeptides, as indicated, were analyzed by immunoblotting with anti-HA or anti-{alpha}-tubulin antibody. CCE, parent ES cells. The extracts were either pretreated with {lambda}-phosphatase (+) or untreated (–). Asterisks indicate nonspecifically reacting bands.

 
These HA-tagged mutated muASK transgene vectors (2nd transgene) and PGK-Hygr drug resistance vector were co-transfected into muASK–/–tg1st ES cells, and surviving cells were selected in the hygromycin-containing medium. Resistant clones were amplified and the expression of HA-muASK mutant polypeptides were confirmed (Fig. 5C). The patterns of mobility-shifts of ASK derivatives were similar to those of COS-7 expressed polypeptides.

Functional dissection of ASK molecule using the ASK-deficient ES cells

These cells were infected with Ad-Cre to excise the 1st transgene (FLAG-muASKwt). N-M-C muASK supported the proliferation as efficiently as the wild-type muASK. This result indicates that the long C-terminal tail of ASK which is found in mammalian and Xenopus ASK/Dbf4 but not in yeast Dbf4/Dfp1 is not essential for the functions of ASK. In contrast, proliferation immediately slowed down in cells expressing {Delta}N, minimum, M-mutants (DY and P) muASK (Fig. 6A). Incorporation of BrdU after Ad-Cre infection was observed with the wild-type and N-M-C polypeptide, but not with minimum, {Delta}N or M mutants (Fig. 6B). Although M mutants, defective in activation of Cdc7, are expected not to support the growth of ASK-deficient cells, it was unexpected that {Delta}N or minimum mutant ASK was not able to support ES cell proliferation in view of their ability to activate Cdc7 both in vitro and in vivo. In yeasts, Dbf4-motif-N was dispensable for the viability of the cells, although the mutants displayed DNA damage sensitivities (Ogino et al. 2001; Fung et al. 2002).



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  		Figure 6  Cell proliferation and DNA synthesis of muASK–/–tg1st ES cells expressing ASK wild-type or mutant polypeptides. (A) The numbers of viable cells were counted at various times (0–4 days) after removal of FLAG-muASK. (B) BrdU incorporation in the same sets of the cells as in (A) was measured as in Figure 4C. (C) muASK+/–tg1st, muASK–/–tg1st or muASK–/–tg1st expressing wild-type or mutant ASK polypeptide as indicated were either mock-infected (–) or Ad-Cre-infected (+). At 48 h after infection, the whole cell extracts were analyzed by immunoblotting with anti-MCM2 antibody (upper) or anti-{alpha}-tubulin (lower).

 
We therefore examined the phosphorylation of the MCM2 protein, a major target of Cdc7, in the cells expressing mutant ASK. MCM2 appears as a series of shifted bands under the wild-type condition. Upon loss of ASK, the fast migrating bands disappear and a single mobility-shifted band appear just below the top band, as was shown in Fig. 4D. The hyper-mobility shift observed in the wild-type background represents phosphorylation caused by Cdc7, since in vitro phosphorylation of the MCM2-4-6-7 complex generated the similar mobility-shift. The shifted band observed after depletion of ASK represents the form of MCM2 phosphorylated by some other kinase. This could be mediated by Cdk (cyclin-dependent kinase), since Cdk phosphorylates MCM2 in vitro, causing the similar mobility-shift (Masai et al. 2000; data not shown). The extensive mobility-shift of MCM2 generated by Cdc7, is maintained in the mutant expressing the wild-type ASK and N-M-C mutant even after Ad-Cre infection. In contrast, in the presence of {Delta}N mutant, the Cdc7-mediated mobility-shift disappeared after excision of the first transgene, suggesting that {Delta}N mutant is not able to support Cdc7-dependent phosphorylation of MCM2 in the cell, presumably required for S phase progression.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we have established a mammalian system in which one can genetically dissect the functions of a gene of interest. This system utilizes a conditional knockout system and is especially useful for factors such as cell cycle regulators which are likely to be essential for cell proliferation. The use of recombinant adenoviruses encoding Cre recombinase permits efficient excision of the transgene, thus inducing the complete loss of the endogenous genes. The mutant genes, the functions of which to be tested, can be readily introduced into the cells as one copy and their functions can be examined.

We have applied this to characterization of ASK encoding an activation subunit for Cdc7 kinase, which is known to be essential for proliferation and DNA replication in mouse ES cells. Recent identification of Drf1/ASKL1 (Montagnoli et al. 2002; Yoshizawa-Sugata et al. 2005), a second activation subunit of human Cdc7, suggested a possibility that the functions of ASK may be redundant with those of Drf1/ASKL1. In fact, both ASK and Drf1/ASKL1 are expressed during S phase. The heterozygous ASK+/– ES cells carrying only one allele of the muASK gene could grow as efficiently as the wild-type ES cells. However, the complete knockout of ASK was obtained only in the presence of the transgene expressing the functional ASK. Therefore, our results clearly demonstrated essential role of ASK for proliferation of mouse ES cells. This is consistent with the serious effect of ASK down-regulation by siRNA on viability and DNA replication in human cultured cells (our unpublished data). Induced inactivation of ASK genes in ES cells lead to almost immediate cessation of cell proliferation as well as DNA synthesis. Cells eventually undergo cell death, as was observed with loss of Cdc7 in ES cells. The cell death is likely to be associated with DNA damages caused by arrested DNA replication forks (data not shown), as was observed in Cdc7 knockout ES cells (Kim et al. 2002, 2003; Kim & Masai 2004).

We were able to functionally dissect mammalian ASK using the established ASK conditional knockout ES cells. ASK/Dbf4 from Xenopus and mammals carry long C-terminal domains which are not conserved. We show that this domain is not required for the essential functions of muASK. As was previously reported for yeast Dbf4/Dfp1, the two conserved motifs, M and C, are essential for binding and activation of Cdc7 as well as for the in vivo functions. One unexpected finding was the requirement of the motif-N, a BRCT-like domain which was previously shown to be involved in interaction with chromatin or other replication factor (Dowell et al. 1994; Pasero et al. 1999; Jares & Blow et al. 2000; Duncker et al. 2002), for the viability of ES cells. The {Delta}N mutant appears to be able to activate Cdc7 as efficiently as the wild-type, as indicated by the autophosphorylation-mediated mobility shift of the ASK molecules as well as by in vitro phosphorylation assays (data not shown). However, the in vivo phosphorylation of MCM2, a critical substrate of Cdc7, is impaired with this mutant. Consistent with this, human minimum ASK lacking motif-N, transfected into the COS-7 cells together with human Cdc7, induced only a very low level of phosphorylation of endogenous MCM2 (Sato et al. 2003). We have also shown that {Delta}N mutant cannot induce the phosphoryation of endogenous MCM4 when transfected into 293T cells with Cdc7 (data not shown), which is observed with the wild-type ASK. These results suggest a possibility that the motif-N serves for recruiting the kinase to its substrate, facilitating its phosphorylation.

The immunoprecipitated minimum ASK-Cdc7 complex can phosphorylate a substrate (an N-terminal polypeptide of MCM2) in vitro in a reaction where an excess substrate is present (Sato et al. 2003). This suggests that Cdc7 in a complex with ASK derivatives lacking motif-N is still capable of recognizing the substrate, albeit with a reduced affinity. The observed requirement of motif-N for MCM phosphorylation in vivo may suggest that the recognition of MCM substrate may be more stringently regulated on the chromatin in the cells.

In fission yeast, cells expressing Dfp1/Him1 lacking motif-N or that carrying point mutations in motif-N can grow but show sensitivity to DNA damaging agents such as HU or MMS (Takeda et al. 1999; Ogino et al. 2001), indicating that the recovery from replication fork blocks is impaired in the motif-N mutants. Therefore, it is also very likely that ES cells expressing {Delta}N ASK are sensitive to replication fork blocks. Thus, the cells’ reduced ability to recover from arrested replication forks may also contribute to the inability of {Delta}N to support the growth, since the ability of the cells to cope with the spontaneous damages which happen during the normal course of S phase in mammalian cells is essential for cell survival (Sonoda et al. 1998).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells

The ES cell line used in this study was CCE28. ES cells were maintained on a layer of mitomycin C-treated feeder cells in the ES media (Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, ES Cell Qualified Nucleosides [1x; Specialty Media] and 0.1 mMß-mercaptoethnol) in the presence of 103 U/mL murine leukemia inhibitory factor (Esgro; Gibco BRL).

Transfection

Transfection of plasmids (targeting and transgene vectors) were performed by electroporation. Twenty µg of linearized plasmid DNA and 107 ES cells were suspended in 1 mL PBS and incubated on ice for 5 min. DNA and ES cell mixture was set on the electroporator (BTX, ECM630) and the electronic pulse was delivered at the parameter of 270 V, 725 {Omega} and 500 µF. The mixture was immediately transferred to the prewarmed ES media and plated on the feeder layer. At 24 h after plating, media was replaced with the ES media containing appropriate antibiotics.

Targeted disruption of the muASK gene

The targeting vector plasmid, in which the 9.8 kb DNA fragment containing the exons 1 and 2 (encoding the first methionine) of muASK was replaced with a DNA fragment containing ß-galactosidase-internal ribosome entry site-neomycin resistance (LacZ-IRES-NeoR.) cassette (Fig. 3A), was linearized and transfected into CCE28 ES cells. Transformants were selected by resistance to G418 at 250 µg/mL for 7–9 days for the presence of the NeoR. genes, and ASK+/– cell lines were cloned.

Southern blot

Genomic DNA was isolated from ES cells using standard protocols and Southern blot analysis was performed using QuikHyb solution (Stratagene).

Construction of a flox transgene (1st transgene) vector

The flox transgene plasmid containing a human EF1{alpha} promoter-driven FLAG tagged wild-type muASK cDNA (FLAG-ASKwt) flanked by the loxP sites (Fig. 3A) was constructed as follows. First, muCdc7 fragment was removed from pEF1{alpha}-loxP-muCdc7-PGKpuroR.-loxP-EGFP (Kim et al. 2002) by SspI digestion followed by self-ligation. FLAG-ASKwt cassette, obtained by NotI digestion of the muASK cDNA fragment inserted into pME18S-FLAG, was inserted at the NotI site of this vector present between the two loxP sites.

Generation of muASK+/– ES cells carrying FLAG-ASKwt transgene and gene conversion

The flox FLAG-ASKwt transgene vector was introduced into the muASK+/– ES cell line by electroporation, and transformants were selected in the presence of 0.9 µg/mL puromycin. Southern blotting and Western blotting were conducted to identify puromycin-resistant clones (muASK+/–tg1st ES) carrying the transgene and expressing FLAG-ASKwt. We then generated muASK–/–tg1st ES cell lines by selecting the survivors in the presence of an increased concentration of G418 (5 mg/mL) for 18–20 days and the clones containing two alleles of neomycin-disrupted ASK were cloned.

Infection of ES cells with adenoviruses

Recombinant adenoviruses, AxCANCre (Ad-Cre; Kanegae et al. 1995), expressing Cre recombinase was amplified on 293 cells and were purified by two rounds of CsCl density centrifugation. They were added at a m.o.i. of 120 to ES cells resuspended in a small volume of ES medium. After incubation for 1 h, cells were diluted with fresh ES medium and were plated on gelatin-coated plates.

Quantification of BrdU incorporation

Ad-Cre infected or non-treated ES cells were seeded on 96 well plate (1000 cells per well). Incorporation of BrdU was measured by using BrdU Cell Proliferation Assay (CALBIOCHEM).

Construction of 2nd transgene vectors

We inserted DNA derived from the oligonucleotide DNAs (EcoRI-HpaI-HAtag-F 5'-AAT TAT GTA CCC ATA CGA CGT CCC AGA CTA CGC TTA CCC ATA CGA CGT CCC AGA CTA CGC TCT GAA TTC CCG GGT CGA CTC GAG CGG CCG C-3' and EcoRI-HpaI-HAtag-R 5'-GCG GCC GCT CGA GTC GAC CCG GGA ATT CAG AGC GTA GTC TGG GAC GTC GTA TGG GTA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAT-3') encoding influenza hemagglutin epitope (HA) tag between the EcoRI and HpaI sites of pEF321-T (Kim et al. 1990) plasmid, a hEF1{alpha} promoter-driven expression vector (located downstream of EF1{alpha} promoter). This EF1{alpha}-HA cassette replaced the SR{alpha} promoter of pME18S plasmid, resulting in pMEF-HA plasmid. Wild-type ASK was amplified with PCR using the muASK-5'primer (5'-ata aga atg cgg ccg caA TGA ACC TCG AGA CCA TGA GGA TCC ACA GCA AAG CA-3')/muASK-3'primer (5'-ata aga atg cgg ccg cCT AAA ATC CAA CGA ATG CAG AAG T-3') set, and the resulting fragment was inserted at the NotI site of pMEF-HA downstream of the HA-tag. {Delta}N ASK was constructed by combining the PCR-amplified and NotI +BssHII-digested DNA fragment (using the muASK-del-N-3'side-F-primer (5'-aca ttt cgc gcg cGT TGA AGA ATT TCT CAG CAA A-3')/muASK-3'primer set) and an annealed duplex DNA (muASK-delt-N-5'ter-F-oligo [5'-ggc cgc aAT GAA CCT CGA GAC CAT GAG GAT CCA CAG CAA AGC Acc tct cg-3'] + muASK-del-N-5'ter-R-oligo [5'-cgc gcG AGA GGT GCT TTG CTG TGG ATC CTC ATG GTC TCG AGG TTC ATt gc-3']) at the NotI site of pMEF-HA. Minimum-ASK was amplified with PCR using Minimum-ASK-F (5'-aag gaa aaa agc ggc cgc aat gGA CAT TCG ATA CTA CAT TGA AC-3')/Minimum-ASK-R (5'-aag gaa aaa agc ggc cgc aCT ATC TTT TCT TTT GAG GTG TGT CTC-3') primer set and the resulting fragment was inserted at NotI site of pMEF-HA. To construct the N-M-C ASK, the long C-terminal tail of the wild-type ASK was removed by the digestion of pMEF-HA-Wild-type ASK with EarI and NotI. M-mut(DY) and M-mut(P) ASK was constructed by PCR-based site-directed mutagenesis. The first PCR was performed using sets of muASK-5'primer/muASK-M-mutant (DY)-R (5'-AAA TGG CCT GGC GCA CCG GTT CAC AGC TTC AAC CTT T-3') and muASK-M-mutant(DY)-F (5'-AAA GGT TGA AGC TGT GAA CCG GTG CGC CAG GCC ATT T-3')/muASK-3'primer or muASK-5'primer/muASK-M-mutant(P)-R (5'-AGC TGA AGG TAA AAT AGC CTG TAG CAC CGG-3') and muASK-M-mutant (P)-F(5'-CCG GTG CTA CAG GCT ATT TTA CCT TCA GCT-3')/muASK-3'primer for M-mut(DY) or for M-mut(P), respectively. For each mutagenesis, the resulting two fragments were isolated and were used for the second PCR in the presence of muASK-5'primer/muASK-3'primer set. The resulting fragments were inserted at the NotI site of pMEF-HA.

Stable expression of ASK mutants in muASK–/–tg1st ES cells

2nd transgene vectors linerarized by FspI and the PGK-Hyg vector carrying the hygromycin resistant gene under the phosphoglycerate kinase gene promoter were cointroduced into muASK–/–tg1st ES cells by electroporation, and transfected cells were cultured in the presence of 200 µg/mL hygromycin for 8 days. Hygromycin-resistant cells were cloned and tested for expression of the HA tagged polypeptides by immunoblot analysis with antibodies against the HA epitope tag.


   Footnotes
 
Communicated by: Fumio Hanaoka

* Correspondence: E-mail: hmasai{at}rinshoken.or.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bork, P., Hofmann, K., Bucher, P., Neuwald, A.F., Altschul, S.F. & Koonin, E.V. (1997) A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 11, 68–76.[Abstract]

Brown, G.W. & Kelly, T.J. (1998) Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast. J. Biol. Chem. 273, 22083–22090.[Abstract/Free Full Text]

Dowell, S.J., Romanowski, P. & Diffley, J.F. (1994) Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science 265, 1243–1246.[Abstract/Free Full Text]

Duncker, B.P., Shimada, K., Tsai-Pflugfelder, M., Pasero, P. & Gasser, S.M. (2002) An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing. Proc. Natl. Acad. Sci. U.S.A. 99, 16087–16092.[Abstract/Free Full Text]

Fung, A.D., Ou, J., Bueler, S. & Brown, G.W. (2002) A conserved domain of Schizosaccharomyces pombe dfp1 (+) is uniquely required for chromosome stability following alkylation damage during S phase. Mol. Cell Biol. 22, 4477–4490.[Abstract/Free Full Text]

Furukohri, A., Sato, N., Masai, H., Arai, K., Sugino, A. & Waga, S. (2003) Identification and characterization of a Xenopus homolog of Dbf4, a regulatory subunit of the Cdc7 protein kinase required for the initiation of DNA replication. J. Biochem. (Tokyo) 134, 447–457.[Abstract/Free Full Text]

Jares, P. & Blow, J.J. (2000) Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading. Genes Dev. 14, 1528–1540.[Abstract/Free Full Text]

Johnston, L.H., Masai, H. & Sugino, A. (1999) First the CDKs, now the DDKs. Trends Cell Biol. 9, 249–252.[CrossRef][Medline]

Kanegae, Y., Lee, G., Sato, Y., et al. (1995) Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res. 23, 3816–3821.[Abstract/Free Full Text]

Kihara, M., Nakai, W., Asano, S., et al. (2000) Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells: its protein kinase activity is regulated by Rad53p. J. Biol. Chem. 275, 35051–35062.[Abstract/Free Full Text]

Kim, D.W., Uetsuki, T., Kaziro, Y., Yamaguchi, N. & Sugano, S. (1990) Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene 91, 217–223.[CrossRef][Medline]

Kim, J.M. & Masai, H. (2004) Genetic dissection of mammalian Cdc7 kinase: cell cycle and developmental roles. Cell Cycle 3, 300–304.[Medline]

Kim, J.M., Nakao, K., Nakamura, K., et al. (2002) Inactivation of Cdc7 kinase in mouse ES cells results in S-phase arrest and p53-dependent cell death. EMBO J. 21, 2168–2179.[CrossRef][Medline]

Kim, J.M., Yamada, M. & Masai, H. (2003) Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development. Mutat. Res. 532, 29–40.[Medline]

Kumagai, H., Sato, N., Yamada, M., et al. (1999) A novel growth- and cell cycle-regulated protein, ASK, activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells. Mol. Cell Biol. 19, 5083–5095.[Abstract/Free Full Text]

Lei, M., Kawasaki, Y., Young, M.R., Kihara, M., Sugino, A. & Tye, B.K. (1997) Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. Genes Dev. 11, 3365–3374.[Abstract/Free Full Text]

Masai, H. & Arai, K. (2000) Dbf4 Motifs: conserved Motifs in Activation Subunits for Cdc7 Kinases Essential for S-Phase. Biochem. Biophys. Res. Commun. 275, 228–232.[CrossRef][Medline]

Masai, H. & Arai, K. (2002) Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell Physiol. 190, 287–296.[CrossRef][Medline]

Masai, H., Matsui, E., You, Z., Ishimi, Y., Tamai, K. & Arai, K. (2000) Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 by Cdks. J. Biol. Chem. 275, 29042–29052.[Abstract/Free Full Text]

Montagnoli, A., Bosotti, R., Villa, F., et al. (2002) Drf1, a novel regulatory subunit for human Cdc7 kinase. EMBO J. 21, 3171–3181.[CrossRef][Medline]

Mortensen, R.M., Conner, D.A., Chao, S., Geisterfer-Lowrance, A.A. & Seidman, J.G. (1992) Production of homozygous mutant ES cells with a single targeting construct. Mol. Cell Biol. 12, 2391–2395.[Abstract/Free Full Text]

Nakamura, T., Nakamura-Kubo, M., Nakamura, T. & Shimoda, C. (2002) Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division. Mol. Cell Biol. 22, 309–320.[Abstract/Free Full Text]

Ogino, K., Takeda, T., Matsui, E., et al. (2001) Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase. J. Biol. Chem. 276, 31376–31387.[Abstract/Free Full Text]

Oshiro, G., Owens, J.C., Shellman, Y., Sclafani, R.A. & Li, J.J. (1999) Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability. Mol. Cell Biol. 19, 4888–4896.[Abstract/Free Full Text]

Pasero, P., Duncker, B.P., Schwob, E. & Gasser, S.M. (1999) A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication. Genes Dev. 13, 2159–2176.[Abstract/Free Full Text]

Saka, Y., Esashi, F., Matsusaka, T., Mochida, S. & Yanagida, M. (1997) Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. Genes Dev. 11, 3387–3400.[Abstract/Free Full Text]

Sato, N., Sato, M., Nakayama, M., Saitoh, R., Arai, K. & Masai, H. (2003) Cell cycle regulation of chromatin binding and nuclear localization of human Cdc7-ASK kinase complex. Genes Cells 8, 451–463.[Abstract]

Sclafani, R.A. (2000) Cdc7p-Dbf4p becomes famous in the cell cycle. J. Cell Sci. 113, 2111–2117.[Abstract]

Sclafani, R.A. & Jackson, A.L. (1994) Cdc7 protein kinase for DNA metabolism comes of age. Mol. Microbiol. 11, 805–810.[Medline]

Sonoda, E., Sasaki, M.S., Buerstedde, J.M., et al. (1998) Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17, 598–608.[CrossRef][Medline]

Takeda, T., Ogino, K., Matsui, E., et al. (1999) A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, plays essential roles in S-phase initiation as well as in S-phase checkpoint control and recovery from DNA damage. Mol. Cell. Biol. 19, 5535–5547.[Abstract/Free Full Text]

Yanow, S.K., Gold, D.A., Yoo, H.Y. & Dunphy, W.G. (2003) Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest. J. Biol. Chem. 278, 41083–41092.[Abstract/Free Full Text]

Yoon, H.J., Loo, S. & Campbell, J.L. (1993) Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle. Mol. Biol. Cell 4, 195–208.[Abstract]

Yoshizawa-Sugata, N., Ishii, A., Taniyama, C., Matsui, E., Arai, K. & Masai, H. (2005) A second human Dbf4/ASK-related protein, Drf1/ASKL1, is required for efficient progression of S and M phases. J. Biol. Chem. 280, 13062–13070.[Abstract/Free Full Text]

Received: 14 February 2005
Accepted: 7 March 2005




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