Cdc7 kinase function is stage-specific in the cell cycle, but total. Cdc7 protein levels ..... activity in various stages of the cell cycle. Relevant. Stage of. Strain.
Vol. 13, No. 5
MOLECULAR AND CELLULAR BIOLOGY, May 1993, p. 2899-2908
0270-7306/93/052899-10$02.00/0 Copyright X 1993, American Society for Microbiology
Cell Cycle Regulation of the Yeast Cdc7 Protein Kinase by Association with the Dbf4 Proteint AIMEE L. JACKSON,1 PAULA M. B. PAHL,' KATHY HARRISON,2 JOHN ROSAMOND,2 AND ROBERT A. SCLAFANI'* Department of Biochemistry, Biophysics, and Genetics, 7he University of Colorado Health Sciences Center, Denver, Colorado 80262,1 and Department of Biochemistry and Molecular Biology, University of Manchester, Manchester M13 9PT, United Kingdom2 Received 25 November 1992/Returned for modification 18 January 1993/Accepted 21 February 1993
Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the GJ/S boundary by using either cultures synchronized with a factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob)-), which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob)-) mutation cannot bypass all events in G1 phase because it fails to suppress temperaturesensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.
cation which is responsible for the activation of the Cdc7 protein kinase at the G1/S transition. One possible mechanism to account for this functional regulation is a stagespecific association with a regulatory subunit, such as a cyclin. Cyclins have been identified as proteins that accumulate periodically in the cell cycle. These proteins serve as regulatory subunits, which bind their catalytic counterparts to result in enzyme activation at a precise point in the cell cycle (4, 10, 12, 14). This form of regulation is seen with the highly conserved p34cdc2 protein kinase, whose protein level remains constant through the cycle but whose kinase activity is regulated by association with specific cyclins at specific times during the cycle (see references 29 and 35 for reviews). One candidate for a regulatory subunit of Cdc7 is the Dbf4 protein. DBF stands for dumbbell former, which reflects the terminal phenotype of mutants with temperature-sensitive mutations in this gene. This terminal phenotype appears as a cell with a single large bud. This dumbbell phenotype is characteristic of DNA replication mutants and is identical to that found for temperature-sensitive (Cdc-) cdc7 mutants. The DBF4 gene was originally identified during a search for replication mutants in S. cerevisiae and, like Cdc7, has been shown to be involved in the initiation rather than the elongation of DNA replication (21). This execution point places the point of function of Dbf4 at the same point in the cell cycle as Cdc7. DBF4 was subsequently shown to be capable of suppressing a temperature-sensitive cdc7 allele when present on a high-copy-number plasmid (22). Likewise, CDC7 present on a high-copy-number plasmid suppresses a dbf4 temperature-sensitive allele. This provides
A fundamental issue in cell biology is the control of the eukaryotic cell cycle. Alterations in cell cycle regulation are implicated in neoplastic cell growth and aberrations in early development (26, 28, 31), indicating that cell cycle control is a key contributor to understanding the basis of cancer and morphogenesis. One approach to understanding the regulation of the cell cycle involves the study of the cell division cycle (CDC) genes of the budding yeast Saccharomyces cerevisiae. Many CDC genes encode proteins which mediate unique and essential roles in the cell cycle. The CDC7 gene of S. cerevisiae is an essential cell division cycle gene which encodes a nuclear serine/threonine protein kinase capable of phosphorylating calf thymus histone Hi in vitro (2, 20, 50). The Cdc7 protein kinase is required for progression through the GJ1S transition of the cell cycle and for the initiation, but not the elongation, of DNA replication in the mitotic cell cycle (17). In addition to its mitotic function, Cdc7 has also been shown to be involved in meiotic recombination (42, 46) and in replication-dependent DNA repair (19). Cdc7 protein function appears to be stagespecific in the mitotic cell cycle, in that Cdc7 has a single execution point occurring exclusively at the G1/S phase transition (17). However, Cdc7 transcript and protein levels remain at a constant level throughout the cell cycle (45). This finding suggests the possibility of a posttranslational modifi* Corresponding author. t This paper is dedicated to Anthony Sclafani and to many others who are victims of cancer. Only continued medical research will help to cure this teffible disease. 2899
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JACKSON ET AL.
genetic evidence for an interaction between these two gene products. Consistent with this possibility, a cdc7dbf4 double mutation is synthetically lethal. Although this synthetic lethality is not evidence for a direct interaction between these two proteins, it suggests that Cdc7 and Dbf4 act in a common pathway. In contrast to CDC7, whose transcript levels remain constant in the cell cycle, Dbf4 is transcriptionally regulated, with maximum transcript levels occurring at the G1/S boundary (7). This peak transcript level occurs at the point in the cell cycle when Cdc7 protein functions, implicating Dbf4 as a potential Cdc7 regulatory factor. Although devoid of a classic cyclin box, the C terminus of the Dbf4 protein contains PEST sequences, which have been implicated in the degradation of proteins that are unstable and susceptible to rapid turnover (30, 37). These preliminary findings strongly suggested the possibility that an interaction between the Dbf4 and Cdc7 proteins may provide the precise regulation of Cdc7 kinase activity at the G1/S boundary and prompted us to pursue genetic and biochemical investigations designed to elucidate this proteinprotein interaction. Our findings demonstrate that the Dbf4 protein does directly interact with Cdc7 to provide the regulatory function necessary for the activation of the Cdc7 protein kinase at the G1/S boundary. We propose a model in which Dbf4 is synthesized and then binds to and activates Cdc7 as a Cdc7-cyclin molecule.
MATERIALS AND METHODS Media, strains, and general methods. A list of strains and plasmids used in this study is displayed in Table 1. All yeast strains (except CTY10-5d) are congenic with A364a (15) and were grown in either yeast extract-peptone-dextrose or supplemented minimal medium. Solid medium contained 2% agar and 2% glucose. Escherichia coli was routinely grown in Luria-Bertani medium supplemented with 50 ,ug of either ampicillin or kanamycin sulfate per ml. Routine cloning procedures were as described previously (44, 45). Yeast genetic methods were as described by our laboratory (44, 45). Immunoprecipitation of Cdc7 from yeast cells. Immunoprecipitation of Cdc7 from yeast cells with a rabbit polyclonal anti-Cdc7 antibody was accomplished essentially as described previously (20, 36). Cdc7 protein kinase assays. Cdc7 protein kinase assays were performed as follows. Cdc7 immune complexes were isolated from log-phase yeast cultures as described above and were used as the source of enzyme for Cdc7-specific Hi kinase assays. Cdc7-specific Hi kinase assays were performed as described previously (18) and as follows. Immune complexes were allowed to equilibrate to the appropriate assay temperature (37°C for wild-type enzyme assays, 22 or 40°C for temperature-sensitive enzyme assays) in Hi kinase buffer (50 mM Tris-HCl [pH 7.5], 15 mM MgCl2, 10 mM NaF, 0.2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride) for 30 min prior to enzyme assay. Reactions were carried out in 30 pl of Hi kinase buffer containing 5 ,Ci of [.y-32P]ATP (NEN; 6,000 Ci/mmol) per ,ul, 250 ,M ATP, 0.5 mg of calf thymus histone Hi per ml, and 1 mM phenylmethylsulfonyl fluoride. Reactions were allowed to proceed at 37°C for 30 min and were terminated by boiling in an equal volume of 2 x Laemmli sample buffer (24). Phosphorylation of histone Hi by Cdc7 was determined by polyacrylamide gel electrophoresis (24) on 10% polyacrylamide gels followed by autoradiography to detect radiolabelled Hi. Quantitation of
phosphorylation was performed on a PhosphorImager (Mo-
MOL. CELL. BIOL. TABLE 1. Yeast strains and plasmids Strain or
Mating type and genotype
plasmid
Strain 130 236 312
410 422 439 516 543 546 573 CTY10-5d
H132 P103 P105 P110 P117 P139 P157 P194 P211 P226 P235 Plasmid pBTM116 pGAD2 pKH122 pKH123 pKH125 pKH126 pKH127 pRH107 pRS292 pRS375
a leu2-3 leu2-112 his7 ural lys2 adel, 2 tyrl cdc7-3 a barl his6 ura3 trpl leu2 cdc7-1 a trpl leu2 ura3 canl his6 barl cdc8-1 a his7 ural cdc4-1 a leu2 his7 lys2 trpl a his7 ura3 lys2 cdc28-1 a leu2 canl pep4 prbl lys2 his7 ura3 regl -501 cdc7-3 (pRH107) a leu2 ura3 trpl his7pep4 prbl cdc28-13 a his3Al ura3 adel,2 canl cyh2 cdc7Al::HIS3
(pRS375)
a regl leu2 ura3 dbf4-1 cdc7-3 (pRH107) a ade2 trpl-901 leu2-3,112 his3-200 gal4 gal80 URA43::lexA op-lacZ a barl his6 ura3 trpl leu2 cdc7-1 (pRS292) a leu2 his7 Iys2 trpl bosl-l a leu2 his7 ural Iys2 adel,2 tyrl cdc7-3 bobl-l a leu2 his7 ura3 adel,2 canl cdc7-3 a leu2 his7 lys2 adel trpl bobl-1 a leu2 his7 cdc7-3 bobl-1 a his7 ura3 trpl dbf4-1 a his7 ura3 lys2 trpl dbf4-1 bobl-1 a leu2 his3 Iys2 ura3 cyh2 bobl-1 cdc7Al::HIS3 a his7 ura3 trpl bobl-1 dbf4Al::URA3 a ura3 Iys2 cyh2 his3 leu2 bobl-1 cdc7Al::HIS3 dbf4Al::URA3
2,u TRP1 LexA-(1-202) 2,u LEU2 GAL4-(768-881) 2,u LEU2 GAL4-(768-881)-CDC7-(1-501) 2,u LEU2 GAL4-(768-881)-CDC7-(453-507) 2,u TRP1 LexA-(1-202)-DBF4-(52-695) 21p LEU2 GAL4-(768-881)-CDC7-(1-452) 2,L TRP1 LexA-(1-202)-DBF4-(52-568) 2,u leu2-d GAL-GAP--*CDC7 2,u LEU2 CDC7 ARS4 CEN6 URA3 CDC7
lecular Dynamics). Units of enzyme activity are reported as units detected by Phosphorlmager quantitation over volume. Cell cycle synchrony. Cell cycle synchrony was performed with AL4Ta barl strains grown to early log phase in either yeast extract-peptone-dextrose or selective minimal medium (44). Cultures were treated with a-factor (100 nM) at 30°C for 3 h to allow cells to accumulate in the G1 phase in response to the a-factor mating pheromone. The extent of arrest was determined by viewing the bud morphology (>95% unbudded and shmoo). Cells were collected by filtration through 82-mm filters (0.45-,um pore size; Millipore) and washed with 5 culture volumes of fresh medium to wash out a-factor. After being washed, cells were resuspended in 2 culture volumes of fresh medium. At 20-min intervals after release from a-factor arrest, an aliquot of cells was removed and used for Cdc7-specific immunoprecipitation as described above. Progression through the cell cycle was monitored by bud morphology and fluorescence-activated cell sorting. Cell cycle arrest with inhibitory drugs and Cdc- mutations. Hydroxyurea (HU) was added to liquid cultures at a final concentration of 10 mg/ml in order to arrest cells in the S phase of the cell cycle. Nocodazole was added to liquid cultures at a final concentration of 10 ,ug/ml in order to arrest cells in the M phase of the cell cycle. Cultures were
VOL. 13, 1993
incubated in the presence of these inhibitory drugs for 3 h at 36°C. For the arrest of cultures by temperature-sensitive cell cycle mutations or a-factor, the cultures were incubated under the blocking conditions (restrictive temperature or a-factor) for 3 h. EMS mutagenesis. Ethylmethane sulfonate (EMS) mutagenesis was measured as follows. The temperature-sensitive (Cdc-) strain 130 was grown to stationary phase in 5 ml of yeast extract-peptone-dextrose medium at 22°C. Cells (1 ml) were collected by centrifugation, washed with 1 ml of 50 mM KH2PO4 (pH 7.0), and resuspended in 1 ml of KH2PO4. EMS was added to a final concentration of 3%. After 1 h with gentle rocking, the EMS was inactivated by the addition of 3.5 ml of 5% Na2S203 with shaking for 10 min. Cells were pelleted and washed with 1 ml of KH2PO4. From this, approximately 1 x 107 cells (40 ,ul) were plated to each of 20 rich-medium plates, and incubated at 36°C to select for revertants to temperature resistance (Cdc+). Dilutions of treated and nontreated cells were incubated at 22°C on rich plates to determine the percentage of survival. Construction of cdc7A1::His3 mutation. The cdc7 deletion mutation cdc7A::His3 was created by use of the one-step gene disruption technique (38). The 680-bp NsiI-BamHI fragment of the CDC7 gene (33) was replaced with a 1.3-kb BamHI-NsiI fragment of the HIS3 gene (44). The resultant plasmid was digested with MluI and EcoRI DNA restriction enzymes to produce a 3.6-kb fragment containing cdc7Al:: HIS3 and flanking yeast chromosomal DNA. This DNA restriction fragment was used to transform diploid strain 299/306 (his3/his3 cdc7-5ICDC7) to His'. An equal number of Cdc- and Cdc+ transformants were found, as expected, because disruption of either the CDC7 or the cdc7-5 gene could occur (45). Sporulation of a Cdc- transformant (strain 540) and dissection of 10 resultant asci produced only two viable spores from each ascus. All viable spores were Hisand Cdc-. This indicates that the cdc7A1::HIS3 is correct, which was verified by genomic Southern analysis (data not shown). The deletion removes about 45% of the CDC7 coding region, including the DNA sequences that are altered in three different cdc7 temperature-sensitive mutations, cdc7-3, -5, and -7 (19). If diploid strain 540 is transformed to Ura+ with the URA3 CDC7 plasmid pRO5 and then sporulated and dissected, His' cells are easily recovered because of cdc7 complementation by pRO5, and all are Ura+. Strain 546 was produced from these asci. Construction of the dbf4Al::UR43 mutation. The dbf4Al:: URA3/DBF4 strains P226 and P235 were created directly by gene replacement, as previously described, by transforming the 2-kb SspI fragment of pKK716 (22) into strains P194 and P222, respectively, and selecting for Ura+. The presence of the deletion was confirmed by Southern blotting to genomic DNA. Construction of plasmids for two-hybrid analysis. Activation domain fusions were based on the vector pGAD2 (8), which carries the coding sequence for residues 768 through 881 of the Gal4 transcription factor followed by a unique BamHI site. pGAD2 was linearized with BamHI, made blunt with Klenow polymerase, and ligated to a 2.0-kb BglII fragment derived from pSJ8 (6) carrying the complete CDC7 coding sequence and which had also been made blunt with Klenow polymerase. The resulting plasmid (pKH122) encodes a fusion protein in which the complete Cdc7 protein is fused C terminal to the Gal4 activation domain via an 8-amino-acid linker. pKH126 was derived from pKH122 by deleting a 650-bp BamHI fragment, which removes the coding region of the C-terminal 55 residues of Cdc7 together
REGULATION OF Cdc7 FUNCTION
2901
with some downstream sequences (33). pKH123 was constructed by cloning this 650-bp BamHI fragment into the BamHI site of pGAD2 to generate an in-frame fusion between Gal4 and the C-terminal domain of Cdc7. DNA-binding domain fusions were based on the vector pBTM116 (kindly provided by Stan Fields), which carries a short polylinker immediately downstream of the lex4 coding sequence. A 2.15-kb XmnI-XbaI fragment carrying sequences coding for residues 52 through 695 (the C terminus) of DBF4 was recovered from pDBF4-4 (7), made blunt with Klenow polymerase, and cloned into the SmaI site of pBTM116. The resulting plasmid, designated pKH125, encodes a protein in which the Dbf4 sequence is fused C terminal to LexA. pKH127 was derived from pKH125 by digesting with StuI and SalI, filling in the ends with Klenow polymerase, and religating. This removes a fragment of 620 bp and truncates the DBF4 coding sequence at amino acid 568. 3-Galactosidase assays. P-Galactosidase activity was measured with sodium dodecyl sulfate-chloroform lysates as described previously (27, 39). RESULTS Cell cycle regulation of Cdc7 protein kinase. As stated previously, Cdc7 protein levels remain constant through the cell cycle, yet Cdc7 function is restricted to the G1/S transition of the mitotic cell cycle. This specific execution point raises the question of the control of Cdc7 function. Among the possibilities for the regulation of Cdc7 function is a posttranslational modification of the enzyme in late G1 phase to activate the enzyme for its function in the G1/S transition. The possibility that Cdc7 protein kinase activity is regulated in the cell cycle was investigated by studying Cdc7 kinase activity in synchronized cultures of strain H132, which contains the 2,u pRS292 plasmid for overexpression of CDC7 (20). The CDC7 overexpression strain was utilized in this study to facilitate detection of Cdc7 protein by immunoblots. Our anti-Cdc7 antibodies are capable of detecting Cdc7 kinase activity without overexpression, but detection of protein by immunoblot still requires overexpression of CDC7 because of the extremely low level of Cdc7 protein present in the cell (45). Similar results have been found by others (50). After a-factor arrest and release as described in Materials and Methods, aliquots of cells (100 ml) were removed at 20-min intervals for the analysis of histone Hi kinase activity in immobilized Cdc7 immunoprecipitates. The doubling time of the H132 strain under the conditions used in this experiment is 90 min. Therefore, the time points analyzed in this experiment encompass approximately 1.5 cycles, making it possible to study Cdc7 activity through more than a single cell cycle. This analysis demonstrated that Cdc7 protein kinase activity is regulated in the cell cycle, while Cdc7 protein levels remain constant (Fig. 1A). Cdc7 kinase activity is low for the first 20 min of the experiment and then increases at 40 min after release from a-factor arrest and returns to its original level at 80 min. At approximately 90 min after release, the cells enter the second cycle, and Cdc7 kinase activity remains low through 100 min. At 120 min after a-factor release, which corresponds to approximately 30 min after the beginning of the second cell cycle, the Cdc7 kinase activity again rises (Fig. 1). Quantitation of the experiment indicates that an 8- to 10-fold increase in Cdc7 kinase activity occurs without a concomitant increase in Cdc7 protein levels. Because the amount of Cdc7 protein
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JACKSON ET AL.
MOL. CELL. BIOL. TABLE 2. Strains and treatments used to study Cdc7 kinase activity in various stages of the cell cycle Strain 6
co Cu
c 0
C :
._
0 c 0
u
t_
o,-
0
50
100
150
Time after alpha factor release (minutes) 1200
100
1000
800 *a u._
600
_
Treatment
400
200
0
50
100
150
Time after alpha factor release (minutes) FIG. 1. Analysis of Cdc7 kinase activity throughout the S. cerevisiae mitotic cell cycle. (A) Comparison of Cdc7 protein concentration (0) and kinase activity (0) in a synchronized yeast culture. Each time point taken after the release from a-factor arrest represents 20 min. The yeast strain used has a doubling time of approximately 90 min under the conditions used in this experiment. Cdc7 protein concentration was quantitated by densitometric scanning of a Cdc7 immunoblot. (B) Comparison of Cdc7 protein kinase activity (0) with the occurrence (percentage) of small buds (U) in a synchronized yeast culture.
does not fluctuate during the experiment (Fig. 1A), the regulation of Cdc7 activity seen in the cell cycle is the result of a posttranslational modification. Furthermore, peak kinase activity occurs with the peak of small budded cells and is a direct reflection of the percentage of small budded cells throughout the remainder of the cell cycle (Fig. 1B). The onset of budding in S. cerevisiae is coincident with the onset of DNA replication in the S phase of the cell cycle. Therefore, the fact that Cdc7 kinase activity peaks with the maximum budding level indicates that Cdc7 protein kinase activity reaches a maximum as cells enter the S phase of the
Stage of
cell
cycle
arrest
0 c
;P-
._
Relevant
genotype
Wild-type CDC7 543 410 312 312 Overexpressed CDC7 H132 H132 H132 H132
cdc28-13 cdc4-1 cdc8-1 cdc8-1 cdc7-1 cdc7-1 cdc7-1 cdc7-1
(CDC7) (CDC7) (CDC7) (CDC7)
360C
G
360C a-Factor, 36°C 360C
G1/S
a-Factor
a-Factor, HU HU Nocodazole
G,
S
G, G1/S S M
cell cycle. This is consistent with the function of Cdc7 in cell cycle progression. To provide further evidence for the point in the cell cycle at which Cdc7 activity reaches a maximum, Cdc7 protein kinase activity in yeast strains blocked at specific stages of the cell cycle by temperature-sensitive mutations, cell cycleinhibitory drugs, or a combination of these methods was analyzed. The strains used in this study and the stages of the cell cycle in which they arrest are shown in Table 2. The cdc28-13 and cdc4-1 strains 543 and 410, respectively, arrest in the G, phase of the cell cycle when incubated at the restrictive temperature of 36°C. The cdc8-1 strain 312 is defective in thymidylate kinase (43), which results in arrest of the cells in the S phase of the cell cycle after incubation at the restrictive temperature. Likewise, treatment of a wildtype CDC7 overexpression strain (H132) with HU, which inhibits ribonucleotide reductase (11), causes the cells to arrest in the S phase of the cell cycle because of the lack of deoxynucleotides. Inhibition of DNA precursor synthesis by HU or inactivated thymidylate kinase with an asynchronous population will produce a culture arrested throughout the S phase, depending upon the position of the cells in the cycle at the time of treatment. Inhibition of DNA precursor synthesis with a yeast culture synchronized in G1 phase by prior treatment with a-factor produces a culture arrested at the G1/S boundary. Treatment of wild-type CDC7-overexpressing cells with nocodazole, a microtubule inhibitor, prevents formation of the spindle apparatus, resulting in arrest of the cells in the M phase of the cell cycle. When calf thymus histone Hi is used as the protein kinase substrate, Cdc7 kinase activity is highest in immunoprecipitates obtained from cells arrested at the G1/S transition (Fig. 2). It should be noted that the CDC7 overexpression strain (Fig. 2B) generates noticeably higher Cdc7 kinase activity at the G1I/S boundary than the wild-type CDC7 strain (Fig. 2A), yet the overproduced Cdc7 protein is regulated in a manner analogous to that of the wild-type protein. Consistent with the results obtained from a-factor synchrony, the results obtained from the cell cycle block experiments demonstrate that Cdc7 activity is regulated in the cell cycle and that this activity directly coincides with the function of Cdc7 in progression through the GJIS transition of the mitotic cell cycle (17). Regulation of Cdc7 protein kinase activity by interaction with Dbf4. One possible mechanism to account for the cell cycle-dependent regulation of Cdc7 kinase activity is interaction with a regulatory subunit such as a cyclin. An abundance of genetic evidence implicates Dbf4 as a potential
REGULATION OF Cdc7 FUNCTION
VOL. 13, 1993 12000-
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0-
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H132 H132 H132 FIG. 2. Analysis of Cdc7 kinase activity detected in each stage of the cell cycle in a wild-type CDC7 strain after stage-specific arrest described in Table 2 (A) and analysis of Cdc7 kinase activity detected in each stage of the cell cycle in a CDC7 overexpression strain (B). H132
cyclin-like molecule for Cdc7 (7, 21, 22). To pursue this possibility, strain 573, a dbf4-1 strain containing the pRH107 plasmid for the overexpression of CDC7 in the presence of galactose (20), was utilized to study the effect of the Dbf4 protein on Cdc7 kinase activity. If Dbf4 does function as a regulatory subunit for Cdc7, then temperature inactivation of Dbf4 will have an inhibitory effect on Cdc7 activity in vitro. Cdc7 immunoprecipitates generated from the CDC7 dbf4 strain grown at the permissive temperature contain a thermolabile kinase activity at 40°C in vitro, whereas Cdc7 immunoprecipitates generated from strain 516, a control DBF4 strain, contain a thermostable kinase activity in vitro (Fig. 3). This is consistent with results we have shown previously (20), which demonstrate that wild-type Cdc7 protein is stable as an enzyme at 40°C. Temperature inactivation of the Dbf4-1 protein from the CDC7 dbf4-1 strain results in a fivefold decrease in Cdc7 kinase activity, while the same treatment produces no decrease in Cdc7 activity from a CDC7 DBF4 strain (Fig. 3). These results clearly
u
Al
CDC7dbf4
FIG. 3. Analysis of the effect of temperature inactivation of the Dbf4-1 protein on Cdc7 protein kinase activity. *, activity at 22°C; X, activity at 40°C. Values represent the average of four experiments.
demonstrate that Cdc7 protein kinase activity is dependent on the presence of an active Dbf4 protein in vitro. In order to determine in a different way whether there is an interaction between the Cdc7 and Dbf4 protein products, protein extracts from strain 236 (cdc7-1) were used to investigate Dbf4 function. A protein extract of strain 573 (dbf4-1) was temperature inactivated as described, such that the Cdc7 protein kinase was rendered inactive because of the inactivation of the Dbf4 protein. Protein extracts of strain 236 generated from either a-factor-arrested or normal cycling cultures were mixed with the inactivated extract from strain 573 in equivalent protein amounts, which was followed by the Cdc7-specific immunoprecipitation and kinase assay. This experiment would serve to determine whether Dbf4 protein from the cdc7-1 strain 236 is able to reactivate Cdc7 kinase activity in the inactivated dbf4-1 extract from strain 573. In addition, this investigation would determine whether the ability of Dbf4 to interact with and activate Cdc7 protein is cell cycle regulated. By using calf thymus histone Hi as the substrate for Cdc7, it was determined that the Dbf4 protein is capable of reactivating Cdc7 protein kinase activity to 65% of its original activity in vitro (Fig. 4). This reactivation demonstrates that Dbf4 protein interacts with Cdc7 protein in vitro to result in activation of the enzyme. In addition, this interaction is cell cycle regulated. A protein extract from the a-factor-arrested DBF4 culture is unable to reactivate Cdc7 kinase activity, whereas a protein extract from a cycling DBF4 culture is capable of this reactivation (Fig. 4). Therefore, the Cdc7-Dbf4 protein-protein interaction occurs after the a-factor arrest point in the G1 phase of the cell cycle. This is consistent with the fact that peak Dbf4 transcript levels occur after the a-factor arrest point but prior to the CDC7 point of the cell cycle (7). It is not yet known whether the cell cycle-regulated activation of Cdc7 by Dbf4 is the result of translational or posttranslational regulation of Dbf4, and experiments are under way to investigate which of these mechanisms accounts for the regulation of the Dbf4-Cdc7
protein-protein interaction. The bob)-) mutation can bypass both cdc7 and dbf4. A suppressor of a cdc7-3 temperature-sensitive mutation was isolated by selecting for growth of strain 130 at the restrictive
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JACKSON ET AL.
MOL. CELL. BIOL.
20000 1
TABLE 4. Tetrad analysis of cdc7A1::HIS3 straina
;.-
Qu
No. of tetrads
His+:His-
4:0 3:1 2:2
2 7 0
2:2 1:2
E
a Table shows results from the cross P180 x 546. A diploid made by
u cX
crossing the strains listed was sporulated, and tetrads were dissected. The relevant genotypes are CDC7Icdc7A1::HIS3 and bobl-lIBOBI.
100l t. ca *S
Viability
*W
lab
422; the diploids sporulated, and the 10 resulting tetrads analyzed according to standard genetic techniques. Asci from 11 of 12 diploids gave a pattern of segregation indicative of an intragenic suppressor, that is, 4+:0- at 36°C, and are probably the result of a reversion of the cdc7-3 allele. Most asci from the diploid produced from revertant 12 gave a pattern of segregation indicative of an extragenic suppressor, that is, 3+:1- at 36°C, and are most likely the result of a mutation in a gene not linked to CDC7 (Table 3). Because a diploid produced by mating revertant 12 (strain P105) with cdc7-3 strain P110 is Cdc-, the suppressor is recessive. When this diploid was sporulated and the tetrads were dissected, only 2+ :2- asci were obtained at 36°C. This indicates that suppression is due to a single chromosomal gene. By similar analysis with three other different cdc7 alleles, cdc7-1, cdc7-4, and cdc7-7 (19), we found that all cdc7 alleles tested are suppressed by this suppressor. Because the suppressor is not allele-specific, we reasoned that it may be bypassing Cdc7 function in the cell cycle. This possibility was tested by determining whether the suppressor could suppress a lethal cdc7A1::HIS3 deletion mutation (Materials and Methods). Strain 546 contains the cdc7Al::
HIS3 mutation and is maintained by the single-copy plasmid pRS375 containing both the URA3 and CDC7 genes. Strain 546 was mated with a ura3 CDC7 strain (P180), which has the suppressor. The diploid was screened for plasmid loss (Ura-), and the resulting CDC7Icdc7A::HIS3 diploid was sporulated. Dissection of these asci produced either three or four viable colonies, representing T (tetratype) or NPD (nonparental ditype) asci, respectively, in which one or two of the colonies were His' (Table 4). Because the lethality caused by the cdc7A1::HIS3 mutation is 100% linked to HIS3, His' colonies can only survive if the suppressor is capable of bypassing the deletion. Similar results were found with a cdc7::URA3 insertion mutation that has been previously described (45). These data indicate that the suppressor can bypass Cdc7 function. However, this suppressor appears to be inefficient, in light of the doubling time of a suppressor strain compared with that of a wild-type strain. A cdc7Abobl-1 strain has a doubling time of 3 h at 30°C and 8 h at 22°C compared with a wild-type strain, which has a doubling time of 2 h at 30°C and 3.5 h at 22°C. We refer to the suppressor gene as BOB1, for bypass of block. We reasoned that the bobl-1 mutation may be bypassing the entire dependent order of events (16, 21) during the G1/S-phase transition of the mitotic cell cycle, specifically the events dependent on the CDC28, CDC4, DBF4, and CDC7 gene products (see Discussion). The cdc28-1 strain 439 and the cdc4-1 strain 410 were individually mated with the bobl-1 strain P117, and tetrad analyses were performed as described above for the cdc7 alleles. In each case, at 36°C only 2+:2- asci were found, indicating that bobl-l cannot suppress the cdc28-1 or cdc4-1 temperature-sensitive mutations (Table 5). However, a cross of bobl-1 strain P103 with dbf4 strain P157 produced 3+:1- and 4+:0- asci at 36°C, demonstrating suppression of the dbf4-1 temperature-sensitive mutation (Table 5). In order to determine whether the suppressor was also bypassing Dbf4 function, the dbf4-1 bobl-1 strain P194 was transformed with a linear piece of DNA in which the 0.3-kb BglII fragment of DBF4 had been replaced with URA3. Ura+ transformants are expected only if the linear piece of DNA has been incorporated into the genome, resulting in a deletion in the DBF4 gene. Furthermore, these transformants
TABLE 3. Tetrad analysis of cdc7-3 revertant strains
TABLE 5. Tetrad analysis of cdc28, cdc4, and dbf4 strains
oI
1
2
4
3
2+3
2+4
FIG. 4. Analysis of the ability of protein extracts from a cdc7DBF4 strain to reactivate Cdc7 kinase activity in a temperatureinactivated CDC7dbf4 strain. Extracts were mixed in equivalent amounts, and Cdc7 complexes were immunoprecipitated with antiCdc7 antibodies. Prior to Cdc7-specific Hi kinase assay, extracts were incubated at the restrictive temperature of 40°C. Cdc7 kinase activity (units per microgram per minute) obtained from each extract was compared with the activity of the CDC7dbf4 strain at the permissive temperature of 22'C (column 1) to gauge reactivation: 1, 573, 22°C; 2, 573, 40'C; 3, 236, a-factor arrest, 40'C; 4, 236, log phase, 40°C. temperature of 36°C after mutagenesis with EMS. Each of 12 revertants (no. 1 through 12) were mated with Cdc+ strain were
No. of tetrads
No. of tetrads (Cdc+:Cdc-) Crossa
4:0
Revertants 1-11 x 422 Revertant 12 x 422 Diploids
made
by crossing
3:1
15 0 the strains listed
0 13 were
sporulated,
2:2
0 2 and tetrads
dissected. Revertants 1 through 12 are revertants of the temperaturesensitive cdc7-3 strain 130 to Cdc+ after treatment with EMS. Strain 422 is wild-type CDC7.
Crossa
439 x P117 410 x P117 P157 x P103
(Cdc+:Cdc-)
Relevant genotype
cdc28-1/CDC28BOB1/bobl-1 cdc4-1/CDC4 BOB1/bobl-1 dbf4-1/DBF4 BOB1/bobl-1
4:0
3:1
2:2
0 0 4
0 0 5
9 19 2
were
a Diploids made by crossing the strains listed were sporulated, and the tetrads were dissected.
REGULATION OF Cdc7 FUNCTION
VOL. 13, 1993
2905
TABLE 6. Transcriptional activation of lacZ by CDC7 and DBF4 hybrids
3-Galactosidase activity (U)
Transformant DNA-binding domain hybrid
Activation domain hybrid
LexA-(1-202)-DBF4-(52-695) LexA-(1-202) LexA-(1-202)-DBF4-(52-695) LexA-(1-202)-DBF4-(52-695) LexA-(1-202)-DBF4-(52-695) LexA-(1-202)-DBF4-(52-568) LexA-(1-202)-DBF4-(52-568) LexA-(1-202)-DBF4-(52-568) LexA-(1-202)-DBF4-(52-568)
GAL4-(768-881) GAL4-(768-881)-CDC7-(1-507) GAL4-(768-881)-CDC7-(1-507) GAILA-(768-881)-CDC7-(453-507) GAIA-(768-881)-CDC7-(1-452) GAL4-(768-881)-CDC7-(1-507) GAILA-(768-881)-CDC7-(453-507) GAL4-(768-881)-CDC7-(1-452)
