Copyright 0 1992 by the Genetics Society of America

Temperature-Sensitivecdc7 Mutations of Saccharomyces cerevisiae Are Suppressed by the DBF4 Gene, Which Is Required for the G1/S Cell Cycle Transition

Kunio Kitada,* LelandH. Johnston: Toshiko Sugino* and Akio Sugino*”

*Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and ?Laboratory of Cell Propagation, National Institutefor Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, England Manuscript received August 13, 1991 Accepted for publication December 20, 1991

ABSTRACT When present on a multicopy plasmid, a gene from a Saccharomyces cereuisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the GI + S phase progression of the cell cycle. DBF4 has an open readingframe encoding 695 amino acid residues and the predicted molecular mass of the gene productis 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and thesporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromo- some replication.

NITIATION of chromosome replication in eukar- determined. The approach we have taken is to isolate I yotes is tightly coordinated in the cell cycle. In the suppressors of the cdc mutations, particularly cdc7, yeast Saccharomyces cerevisiae, several cell-division- because the CDC7 geneproduct is believed to be cycle (cdc) mutations that block initiation of chromo- required at the last step prior to initiation of chro- some replication have been characterized [see CAMP- mosome replication (CAMPBELL1986; PRINCLE and BELL (1 986), PRINCLE and HARTWELL (1981) and HARTWELL198 1 ; NEWLON1988). A genesuppressing NEWLON (1988) forreview], for example, cdc28, -4, - cdc7 mutations was isolated froma yeast genomic 6 and -7. The execution points of the corresponding library constructed with a high copy number vector. genes are unique during the cell cycle and the cell Restriction mapping and complementation analyses must progress through a conditional series of steps with the isolated gene revealed that it is identical to (i.e., CDC28 + CDC4 + CDC7) before initiation of DBF4, which we isolated independently(CHAPMAN chromosome replication can occur (CAMPBELL1986; and JOHNST~N 1989). dbf4, as well as dbfl, -2 and -3 PRINCLE and HARTWELL 198 1; NEWLONHERE- 1988; mutants, are defective in DNA synthesis and exhibit FORD and HARTWELL 1974). adumbbell terminal morphology atthe restrictive The CDC28, -4, -6 and -7 genes have been cloned temperature (JOHNSTON and THOMAS1982a,b). The and biochemical and genetic studies (LORINCZand execution point of DBF4 in the cell cycle was mapped REED1984; PATTERSONet al. 1986) have revealed after that of CDC4, but before that of CDC7 (JOHN- that the CDC28 and CDC7 polypeptides have protein STON and THOMAS1982b). Inthis report, we describe kinase activity, implicating protein phosphorylation in the nucleotide sequence of DBF4 and genetic analyses the GI + S phase progression of the cell cycle. The of CDC7 and DBF4. Based on these data, we suggest predicted amino acid sequence of the CDC6 gene has thatboth gene products must interact to facilitate a nucleoside triphosphate bindingconsensus sequence initiation of yeast chromosome replication. (LISZIEWICZet al. 1988; ZHOU, HUANCand JONC 1989) and CDC4 exhibits some amino acid sequence MATERIALS AND METHODS similarity to the ets oncogene (PETERSONet al. 1984). While considerable informationis now available about Strains and media: Yeast strains used in this study are listed in Table 1. KKY228 was constructed by gene replace- these genes, their roles in replication and possible ment (ROTHSTEIN1983) with the cdc7-1 gene on pKK319. interactions among their gene productshave not been KKY701 (dbf4::URA3/DBF4) and KKY711 (cdc7::LEU2/ CDC7) diploid strains were constructed by replacing the ’ To whom correspondence should be addressed. wild-type chromosomal gene with the dbf4::URA3 gene on

(knetics 131: 21-29 (May, 1992) 22 K. Kitada et al.

TABLE I Yeast strains

~~ Strain Genotype Source

208 MATa cdc7-1 ura3-52 leu2-3, 112 PATTERSONet al. (1986) 136 MATa cdc7-2 ura3-52 leu2-3, I12 PATTERSONet al. (1986) 209 MATa cdc7-3 ura3-52 leu2-3,I1 2 PATTERSONet al. (1986) 142 MATa cdc7-4 ura3-52 leu2-3, I12 PATTERSONet al. (1986) L 128-2D MATa dbf4-I ura3 trpl CHAPMANand JOHNSTON (1989) L128-1 D MATa dbf4-l leu2-3, -112 ura3-52 From cross L128-2D with CG379 (DYKSTRAet al. 1991) L202-1A MATa dbf4-2 ura? leu2 trpl CHAPMANand JOHNSTON (1989) KKY228 MATa cdc7-l ura3-52 leu2 trpl-289 This study KKY401-1OB MATa dbf4-1 ura3 trpl leu2 This study KKY700 MATa/MATa ura3-58/ura3-58 Our laboratory stock leu2-3, -112/leu2-3, -112 trpl- 289/trpl-289 KKY7Ol MATaIMATa DBF4/dbf4::URA3 This study ura3-52/ura3-52 leu2-3, 1121 leu2-3, 112 trpI-289/trpl-289 KKY701-13AC MATO dbf4::URA3 ura3-52 leu2-3, This study 112 trpl-289 [pKK821] KKY711 MATaIMATa CDC7/cdc7::LEU2 This study ura3-52/ura3-52 leu2-3, 1121 leu2-3, 112 trpl-289/trpl-289 KKY724 MATalMATa CDC7/cdc7-2 dbf4-I/ 136 X L128-1D DBF4 ura3lura3 leu2/leu2 KKY725 MATalMATa cdc7-I/CDC7 DBF4/ 208 X KKY401-lOB dbf4-1 ura3lura3 leu2/leu2 KKY727 MATaIMATa cdc7-4/CDC7 DBF4I 142 X KKY401-1OB dbf4-1 ura31ura3 leu2/leu2 KKY743 MATa dbf4-3 ura3 leu2 trpl This study KKY744 MATa dbf4-4 ura? leu2 trpl This study KKY745 MATa dbf4-5 ura3 leu2 trpl This study

pKK716 and the cdc7::LEU2 gene on pKK7 10, respectively. yeast cells, total DNA was extracted as described by SHER- These constructions were verified by Southern blotting of MAN, FINKand HICKS(1986) and used for E. coli transfor- chromosomal DNA digested with the appropriaterestriction mation. In most cases, the electrophoration method (Bio- enzyme(s) followedby DNA-DNA hybridization. Yeast cells Rad Laboratories) was used for E. coli transformation. were grown in supplemented minimal medium or YPAD DNA manipulations and genetic techniques: Cloning, (SHERMAN,FINK and HICKS1986). To isolate Ura- yeast preparing plasmid DNA from E. coli and Southern hybridi- cells (BOEKEet al. 1987), 1 mg/ml of 5-fluoroorotic acid zationwere performed as described (MANIATIS,FRITSCH (FOA) (Sigma Biochemicals)and 20 pg/ml uracilwere added and SAMBROOK1982). DNA sequence analysis was carried in the medium. Escherichiacoli strains DH5a (MANIATIS, out by the dideoxy chain-termination method (SANGER, FRITSCHand SAMBROOK1982) andJM 101 (MESSING,CREA NICKLENand COULSON1977) using double-stranded DNA and SEEBERG1981) were used to prepare plasmids. and Sequenase (United States Biochemical)and both strands Plasmid DNA: The S. cerevisiae genomic library in the of the DNA were at least once sequenced. Yeast transfor- YEp 21 3 vector (SHERMAN,FINK and HICKS1986) has been mation was carried out by using lithium acetate methods described (HASEGAWA,SAKAI and SUCINO1989). For sub- (ITOet al. 1983), and otheryeast genetic manipulations were cloning both CDC7 and DBM, either YCplac22, YCplac33, as described by SHERMAN,FINK and HICKS(1986). YCplac 1 11, YEplac 1 12,YEplac 195 or YEplac 181 (GIETZ Isolation of new temperaturesensitivedbf4 mutations: and SUGINO 1988) wereused. pKK716 containing New temperature-sensitive alleles of dbf4 mutation (dbf4-3, dbf4::URA3 was constructed by replacing the 0.3-kb BglII -4 and -5) (Table 1) were isolated by the plasmid-shuffling fragment of DBF4 (Figure 1) with the 1.1-kb URA3 gene. method described previously (ARAKIet al. 1991). We do not pKK7 10 containing cdc7::LEU2 was constructed by replac- know whether dbf4-3 and -4 are different from the alleles ing the 0.4-kb EcoRV fragment of CDC7 (PATTERSONet al. previously isolated (JOHNSTON and THOMAS1982a,b), but 1986) with the 2.2-kb LEU2 gene. pUC18 containing the dbf4-5 is a new allele (see the text). 3.3-kb BglII-EcoRI cdc7-Z gene (pKK319) was identified Other materials: A Multiprime labeling kit (Amersham) from the library constructed with the strain 208 (cdc7-Z) was used to labelDNA probes. 5’-Endof the synthetic genomic DNA digested with BglII and EcoRI enzymes by oligonucleotide was labeled with [y-”PP]ATP and T4 poly- colony hybridization to the 5’-32P-labeled synthetic oligo- nucleotide kinaseas published elsewhere (MANIATIS, nucleotide probe corresponding to a part of CDC7 (”’ATG- FRITSCHand SAMBROOK1982). All restriction enzymes, E. ACAAGCAAAACGAAGAATATCGATGATATACCT~’, coli Klenow DNA polymerase I, T4 polynucleotide kinase, from the nucleotide number 1 to 36 in the coding region) T4 DNAligase, and S1 nucleasewere from IBI, New (PATTERSONet al. 1986). To recover plasmid DNA from England BioLab, BRL, Boehringer Mannheim, Amersham, Suppressor of cdc7 Mutations 23 A suppmsslon of EwW EcoM Xhd Xhol pa1 SnaB1 SnaBI Sphl cde7" I I1 I I I II I I I I + Ylul Sstl AW Awl -1 kb + + FIGURE1.-Restriction map of the gene sup pressing cdc7-1 and construction of a deletion mu- tant. (A) Fach DNA fragmentshown by aheavy line was clonedinto the multicopy yeast vector YEplac 195 (GIETZ and SUGINO 1988) and intro- duced into KKY228 cells by transformation. The transformants were placed at 33" for 3 days and cell growth was examined. The lowest line shows the DBM DNA fragmentisolated by CHAPMAN and JOHNSTON(1989). (B). To constructa dele- tion/insertion mutation of DBF4, the 0.3-kb Bglll fragment was replaced with 1.1-kb BamHl URA3 DNA. The openarrow indicates theORF of DBF4.

77- Adbf4 ::URA3 bJ bJ or United States Biochemical and used as recommended by transformation using the plasmid DNA from T33 cells the supplier. showed that anEcoRI-PstI fragment was the minimum fragment able to suppress temperature-sensitive cell RESULTS growth phenotype of cdc7-1 mutation (Figure 1). The restriction map of this fragment with GcoRV, PstI, Suppression of cdc7-1 by a gene on a multicopy SstI, BcoRI, AvaI and CZaI was identical to that of plasmid: Strain KKY228 (cdc7-I) is temperature-sen- DBF4, which was isolated by complementation of dbf4- sitive for growth on a YPAD plate above at 33". To 1 (CHAPMANand JOHNSTON 1989). Furthermore, a isolate muticopy suppressors which is able to rescue a partialnucleotide sequence of the EcoRI-PstI frag- temperature-sensitive cdc7 mutant phenotype, cdc7-1 ment was identical with part of DBF4 (CHAPMANand cells were transformed with a yeast genomic DNA JOHNSTON 1989). We therefore tested suppression of library in YEp213 and incubated at 24"on Leu- cdc7-1 by DBF4. As shown in Table 2 and Figure 1, plates for two days and at 33" for 5 days. Among DBF4 allowed temperature-sensitive cdc7-1 cells to about 160,000 transformants grown at 24", 18 also grow on either a Leu- or YPAD plate at 33". Fur- grew at 33". When they werestreaked on a fresh plate, two, T33 and T34, formed much smaller colo- thermore, theplasmid (pKK616) from T33 cells com- nies at 33" than the others, but their colony size was plemented dbf4-1 and dbf4-2 as well as didthe authen- uniform. Total DNA was extracted from the 18 trans- tic DBF4 gene (CHAPMANand JOHNSTON 1989). We formant cells, electrophoresed, transferred to a nitro- conclude from these results that the gene on either cellulose filter, and hybridized with radioactive DNA YEp213 or YRpl2 plasmid suppressing cdc7-1 is fragments of CDC7 and pBR322. Plasmid DNA from DBF4. The suppression of cdc7-1 by DBF4 was tem- all transformantsexcept T33 and T34 hybridized perature-dependent. The cdc7-1 cells having DBF4 with both fragments, indicating that they contained formed much smaller colony at 35" than at 33" and CDC7. Thus, they were notcharacterized further. the cells did not formcolony at 37". At those temper- DNA from T33 and T34 hybridized to 32P-labeled atures, however, cdc7-1 cells containingauthentic pBR322 DNA but not to the radioactive CDC7 DNA CDC7 on a plasmid formed the same size of colony as (data not shown), indicating that T33 and T34 cells wild-type cells. harbored a plasmid containing a DNA insert other To test the effect of gene dosage onthe suppression than CDC7. of cdc7-1, DBF4 was transferred to a single-copy plas- Restriction enzyme analysis showed that the plas- mid containing a CGN4 sequence. As shown in Figure mids from both T33 and T34 cells possessed an iden- 2, cdc7-1 cells having DBF4 on a CEN4 plasmid did tical 6.2-kb insert. Deletion analysis followed by re- not form colony on a YPAD plate at 33". Thus, the 24 K. Kitada et al. TABLE 2 cdc7- 7 DBF4 on a multicopy plasmid suppresses cdc7 mutations A B Plasmid Plasmids

Ter11peraturr pKK6 I6 pKK709 Kvlvwnt xrwtype ("C) (DBF4) YEplx 195 (CDC7)

rdr7- I 33 +' - ++D 35 +/- - ++ 37 - - ++ rdc7-2 33 + - ++ 35 +/- - ++ 37 - - ++ pKK709 (CDCT) cdc7-3 33 + + ++ 35 +/-c +I-< ++ 3 7 - - ++ FIGURE:2.--I)HF4 on a rnulticopy plasmid suppresses the tem- perature-sensitive growth phenotype of cdc7 mutations. KKY288 cdr7-4 33 + - ++ (cdc7-1) cells transformed by the indicated plasmid DNA were 35 +/- - ++ patched on Leu- plates and incubated at 24" for 4 days. Then, the - - 37 ++ cells grown were replica-plated on a YPAD plate and incubated at Acdc7" 25 - - ++ 33" for .5 clays andphotographed. The pKK821plasmid was constructed from the plasmid YCplac22 containing CEN4 (GIETZ KKY'L'LH cells were transformed to Ura' with pKK616 (DEF4 in and SUGINO1988) by inserting the EamHI-PstI fragment of DEF4 YEpIxl95). YEplac19.5, or pKK709 (CDC7 in YEplac195). The (Figure I). A and B represent two independent transformants of resulting transformants were streaked on YPAD plates, incubated at the indicated temperatures for more than 3 days and their colony KKY228 with each plasmid. li)rmation WIS examined. + indicates that the cells formed colonies on the plate at the Leu+:Leu- segregation. These results clearly demon- indicated temperature and - indicates that the cells did not form strate that DBF4 does not suppressthe CDC7 deletion colonies at that temperature. cdc7 mutant cells harboring pKK709 formed much larger col- mutation but can suppress all other cdc7 temperature- onies than the cells harboring pKK6 16. sensitive alleles so far tested. ' Although cells harboring either pKK6 16 or YEplac195 formed Mitosis-specific cdc7 suppression by DBF4: It is colonies at 35". the colony size of cells containing pKK616 was ~nuchlarger than that with YEplac195. None of the cells formed known that CDC7 is also required during meiosis, not cdonies at temperatures higher than 35". for initiation of premeiotic DNA replication but for Due to the lethality of cdc7A::LEU2, the KKY7 11 diploid cells premeiotic homologous recombination (SCHILDand liarboring pKK709 were sporulated and microdissected and a Ura' 1x11' spore was isolated. This cell, KKY71 I-[pCDC7-URA3], was BYERS 1978). We therefore investigated whether f'urther transfornled with pDBF4-TRPI plasmid. The resulting cells DBF4 suppresses sporulation defect phenotypeof cdc7 were plated on FOA plates to test cell growth; no cells grew. mutations at the restrictive temperatures. In contrast DBF4 copy number is important for suppression. to the above results, the sporulation defect of cdc7 mutationsat the restrictiontemperatures was not cdc7A mutation is not suppressed by DBF4: We suppressed by DBF4 on a multicopy plasmid (Table investigated whether the suppression of cdc7-1 by 3). The failure of suppression by DBF4 is not due to DBF4 is allele-specific. As shown in Table 2, all tem- inability of expression of DBF4 in meiosis, since DBF4 perature-sensitive alleles of cdc7 mutations (cdc7-I, 7- is required for meiosis (Table 3) and it was confirmed 2, 7--3, and 7-4)were suppressed to varying degrees that the DBF4 gene complements sporulation defect by DBF4 on a multicopy plasmid. To test whether a of dbf4-1 mutation (Table 3). We therefore conclude cdc7A mutation is suppressed by DBF4, the heterozy- that suppression of cdc7 mutations by DBF4 is mitosis- gous diploid strain KKY711 (cdc7::LEU2/CDC7)with specific. the DBF4 geneon a multicopy plasmid containing Allele specific, mitotic suppression of dbf4 by URA3 (pKK6 16) was sporulated,tetrads were dis- CDC7: As shown in Table 4, CDC7 on a high copy sected, andthe resulting spores were incubated at number plasmid suppressed the dbf4 mutations by an 24". We obtained only two viable spores from each allele-specific manner. CDC7 suppressed the temper- tetrad (total of 27 tetrads analyzed). All viable spores ature-sensitive dbf4-I, -2, -3and -4 mutations but not were Leu- and 55% were Ura+. More than 90% of the temperature-sensitive dbf4-5 and dbf4 deletion the inviable spores exhibited a dumbbell morphology mutations. Furthermore, as observed with DBF4 on a YPAD plate as observed in all DNA replication suppression of cdc7 mutations, suppression by CDC7 mutants incubated at a nonpermissive temperature. occurs only witha multicopy plasmid (data notshown). In contrast, the same diploid strain with CDC7 on a dbf4 suppression by CDC7 was mitotic specificlike high copy number plasmid producedfour viable cdc7 suppression by DBF4 and CDC7 could not sup- spores in 65% of the dissected tetrads (total of 24 press sporulation defect of dbf4-f mutation (Table 3). tetrads were analyzed), and these spores exhibited2:2 Incompatibility of dbf4 and cdc7 mutations: To Suppressor of cdc7 Mutations 25 TABLE 3 TABLE 4 Suppression of meiotic defect of either dbf4-1 or cde7-1 CDC7 on a multicopy plasmid suppresses the temperature- mutation sensitive dbf4 mutations

Sporulation efficiency (% asci) with Plasmids plasmid: Relevant Temperature pKK602Temperature pKK603 genotype ("C) DBF4YEp in CDC7YEPYEp in Relevant genotype ("C) (CDC7) YEp213 (DBF4) dbf4-lldbf4-1 25 40.2 f 9.3 37.2 f 2.1 32.2 f 2.7 dbf4-1 35 + - + 35 25.8 f 7.3 4.2 & 1.6 8.7 f 2.2 37 + - + cdc7-llcdc7-1 25 26.2 f 1.4 25.7 f 3.1 21.5 f 5.0 dbf4-2 35 + - + 32 2.0 f 0.7 13.9 & 1.7 2.0 f 1.2 37 + - + Diploid cellsL128-2D/KKY401-10B (MATaIMATa dbf4-lldbf4- dbf4-3 35 + - + 1 ura3/ura3 trplltrpl LEU2 /1eu2) and 208/KKY228 (MATal 37 + - + MATa cdc7-1/cdc7-1 ura?-.52/ura?-52 -1leu2-3,12/leu2 TRPl ltrpl- dbf4-4 35 + - + 28Y) were transformed with either pKK709 (CDC7), pKK616 - (DFB4)or YEplacl95 and the transformants were grown in SPS 37 + + medium to 5 X IO' cells/ml and the cells were resuspended to the dbf4-5 33 - - + acetate medium (SUGINO,NITISS and RESNICK1988). Sporulation 35 - - + efficiency (% asci) was measured as published (SCHILDand BYERS 37 - - + 1978) atthe indicated temperatures. Ad bf4 a 25 - - + obtaina cdc7-1dbf4-1 doublemutant, the diploid KK401-IOB (dbf4-1),L202-1A (dbf4-2), KKY743 (dbf4-?), strain KKY725 (ura3-52/ura3-52 dbf4-1IDBF4 CDC7/ KKY744 (dbf4-4)and KKY745 (dbf4-5)cells were transformed to cdc7-1) was sporulated at 24".To avoid any possibility Leu+ with either pKK602 (CDC7 in YEp213) (SHERMAN,FINK and HICKS1986), YEp213 or pKK603 (DBF4in YEp213). The growth of incompatibility of dbf4 with cdc7 mutation,the of the transformants was examined as in Table 2. + indicates that strain was first transformed with pKK709 containing dbf4 mutant cells grew at the indicated temperatures on both a CDC7 and URA3 before sporulation. The resultant YPAD and synthetic complete medium (SD) plates without leucine, while - indicates that the cells did notgrow under these conditions. tetrads were dissected and incubated at 24". Tetrads a Due to the lethality of dbf4k:URA3, KK701 diploid cells were giving four viable Ura+ colonies were tested whether transformed with plasmidpKK602 and sporulated and theresulting they are able to grow without the plasmid pKK709 tetrads were dissected. Only 2 viable spores were obtained from each tetrads dissected (total 24 tetrads analyzed). On the other (CDC7)by incubating on FOA plates at 24 ". From 39 hand, KKY701 cells harboring pKK603 gave 4 viable spores from tetrads analyzed, 7, 22 and 10 tetrads exhibited 4, 3, 12 (50%) tetrads out of 24 tetrads dissected. These exhibited 2:2 and 2 spores viable, respectively. Each tetrad exhibit- segregation for Ura+ and 4:O for Leu+. ing sporesgrowing on aFOA plate was further 2 al. 1988), the cells were plated on YPAD plates and examined by crossing with the strain carrying either incubated at 24". When a daughter cell emerged, it cdc7-Z or dbf4-1 mutation. The two viable colonies on was separated from the mother cell by micromanipu- a FOA plate had neither cdc7-1 nor dbf4-Z mutation, lation and incubated at 24". Analysis of 642 such cell whereas the two inviable colonies on a FOA plate had divisions revealed 11 loss divisions, that is, divisions in both cdc7-Z and dbf4-Z mutations. From this result, which either the mother or daughter cell failed to we conclude that a strain containing both cdc7-1 and receive a plasmid molecule and yielded microcolonies dbf4-Z mutations is inviable without the CDC7 gene. containing an average of 4.7 (range 2-10) inviable Similar results were obtained with combinations in- cells.Most of these inviable cells exhibited a large volving dbf4-1 and dbf4-2 with cdc7-2, cdc7-3 or cdc7- dumbbell shape, typical of DNA replication mutants 4. Thus, cdc7 and dbj4 mutations are incompatible. at a restrictive temperature (PRINGLEand HARTWELL Division potential conferredby amount of DBF4 1981). At the time of cytokinesis, therefore, a cell gene product: The amount of DBF4 transcript fluc- whichlost the plasmid either has sufficient DBF4 tuates during the cellcycle and peaks at the GI/S protein to divide at least twice. Alternatively, a cell boundary similarly to other genes involved in DNA synthesizes enough DBF4 proteinfrom remaining replication UOHNSTONet al. 1990). This suggests that mRNA although the protein turnsover quickly. This the amount of DBF4 product also increases late in G1 result is significantly differentfrom similar results phase and that the newly synthesized DBF4 protein is obtained with CDC7 (SCLAFANIet al. 1988), where at required for initiation of chromosome replication. To least 8 divisions occurred after loss of the plasmid. examine this possibility, the amount ofDBFI product DBF4 is essential for mitotic cellgrowth Isolation was estimated cytologically by examining division po- of temperature-sensitivemutation does not ensure tential after removing DBF4 from dbf4::URA3 cells. essentiality of its gene. Therefore, we tested whether A dbf4::URA3 strain carrying the DBF4 gene on a DBFI is required for mitotic cell growth. To dothis, plasmid containing CEN4 was constructed (KKY70 1- one chromosomal DBFI gene in a diploid cell was 13AC, Table 1).As previously described (SCLAFANIet disrupted asshown in Figure 1B. The resultant diploid 26 K. Kitada et al.

-360 TATCTCTTTGCATCTAATTCCTCATTACCTTTTTTATTTTTATTTCCTCTTATCGGTCCTCTTCGAACGCCTAAGT~TTTTAATATTTTA~C~A~AAGTAAATGGTTTTTTTTTT~AA -240 ~CTTAGGCCTCGATCTTTGCG~G~TAAAGAAATAACGATAATAATAACGGTAATGGCTCAGGCCAATAGT~C~T~TAAAAGGCTGCAT~CCTCTATACTCAGCC~G -120 TGTAGATTCCTTTAGATCTGTCCTCGCTTGCA~CTAGTTATCTCCTCAGAATTCAGTTTGARTCTGAAAGGCATTT~TTCTGCTTT~GTCCTCATA~TAGAAAAG~GAAGAAA 1 ATGGTTTCCCTCCAACGAAAATGATAATAAGGCCTCTCCGTTAAT MVSPTKMIIRSPLKETDTNLKHNNGIAASTTAAGHLNVFS 40 121 80 241 RARSIEGAVQVSKGTGLKNVEPRVTPKELLE~WQT~NW~K~KI~M120 361 AAAAGAGATTCTCTAGATGCATTTACTTTGACATTACT~~TGTAGAGATGAATACATATMTAAGTCCAAGATGGACAAACGCAGAGATTTAT~GGG~TCTTACATTAAATACA KRDSRIYFDITDDVEMNTYNKSKMDKRRDLLKRGFLTLNT 160 481 CARATAACTCAATTTTTTGACACTACTGTCACAATAGTTAGTTATCACMGAAGAAGGCCTCTGTTGAGAACATATATTTACT~GATACCGACATTTTATCGAG~T~GTACATG QITQFFDTTVTIVITRRRSVENIYLLKDTDILSRAKKKYM 200 601 AAAGTTTGGAGTTACGAAAAGGCTGCCAGATTTCTGAAAAATCTTGATGTTGATTTGGATCATT~AGCAAGACTAAACCTCT~TTCTTTAGC~~CACA~GTCCMTCTTCTACAC KVWSYEKAARFLKNLDVDLDHLSKTKSASLAAPTLSNLLH 240 721 AATGAAAAATTATATGGACCAACGGATAGAGACCCCAGAACT~GA~GATATTCACTACTTTAAATATCTAGATCTCATGTATACCTTTATGA~TA~~TT~CCCCATMTA NEKLYGPTDRDPRTKRGDIHYFKYPHVYLYDLWQTWAPII 280 841 ACTTTGGAATGGAAACCTCAGAACTAACAAACTTAGACGAACTACCTTACCCAATATTG~TAGGTTCATCCTCGGAAGA~C~TTTTATAG~GAT~GGAATTATGACGAAAGTTCT TLEWKPQELTNLDELPYPILKIGSFGRCPFIGDRNYDESS 320 961 TATAAGCGCGTAGTAAAGAGATACTCGAGAGACAAAGCAAAC~TATGCACTGCAACTTCGTGCTCTATTTCTAGATAATAGTTATCAT~CGACACCTTAC~~T~GTCATCAGTTAATGAT YKRVVKRYSRDKANKKYALQLRALeQYHADTLLNTSSVND360 1081 CARACGAAAAACCTAATATTCATACCTCTAGATACACATGCAACGATTCTACCAA~GCTTC~TGGATGCAAGAAAA~~TTTTGA~GA~~TTAAAGAAGACGGATGAT QTKNLIFIPHTCNDSTKSFKKWMQEKAKNFEKTELKKTDD 400 1201 AGCGCAGTTCAAGATGTTCGTAATGAACATGCTGACCAAACCGAT~CAAAAGAGCCTCCGTTGAAAGAAG~~TAAGC~~TATAGCA~~~~TAAGTACCCA SAVQDVRNEHADQTDETKEPPLKEEKENKRSIAEESNKYP 440 1321 CAGCGAAAAGAGCTGGCTGCCACACCAAAACTAAACCACCTCCAGTATTAGCTACTTT~CMG~AAGAAACTGAA~T~~ATGATTTGTGCACTT~~CAAAGTCACGTCAG QRKELAATPKLNHPVLATFARQETEEVPDDLCTLKTKSRQ 480 1441 GCATTTGAAATCAAAGCAAGTGGGT~ACATCAATCTAATGATGTGGCAACCTCTTTTGGCAA~TT~GCCCAACAAGAGCAA~~GTCATGAGTAAGAACATGAAGTCATTAATAGTGTAGA AFEIKASGAHQSNDVATSFGNGLGPTRASVMSKNMKSLSR 520 1561 CTAATGGTTGATAGAAAGCTGGGAGTAAAGCAGACAAATG~TAAC~TTATACAGCCACTATAGCAACTACT~TGAAACATCAAAGG~TAGACACAGATTAGATTTTMT LMVDRKLGVKQTNGNNKNYTATIATTAETSKENRHRLDFN 560 1681 GCTTTGAAAAAAGACGAAGCCCCTTCTAGATGAAAGAGAC~CAAAGATAGTGCTGTACACTTAGAAACTAATA~GCCCCAGAATTTCCCTAAGGTAGCTACCAAATCAGTCTCCGCA~C ALKKDEAPSKETGKDSAVHLETNRKPQNFPKVATKSVSAD 600 1801 TCCAAAGTTCATAATGACATCAAGATAACAACCACAGAATCTCCAACAGCACCTCGAAGAAATCAACTTCCACAAACGTCACCTTACATTTTAACGCACAGACA~ACAGACAGCACAGCCG SKVHNDIKITTTESPTASKKSTSTNVTLHFNAQTAQTAQP 640 1921 GTGAAGAAAGAAACGGTATTCCGGATACTGTGAAAT VKKETVKNSGYC~ENC~~RVKYESLEQHI~VS-EKH~LSFAENDLN680 2041 TTTGAGGCTATTGACTCGTTAATTGAAAATCTCAGATTCCTCAAATATAGGGACACGACGT~GTGCAGTAGCTTTTAGTGAT~TC~TAGTATTGTCCTCCG~TCCATT~~TC FEAIDSLIENLRFQI' 1695) 2161 GGAACAAAAAAGCTATCAACGGCAATGTTATTGAATCACTTTCTCATTCAC~TTGTTACTTTCTTGCTATTGACTTAACTC~ATTTACTCGTCCATATATTATCMT~ATATATAT 2281 ACATATACATATATATATTATCATCTAGATTAGATTAATTACCGTTCATATCAT~TCATCCTCTGAACAAATMTGACCTCGAGTACCCACCGCGGTTAATGTC~AGT~CCTCATTT~AC~GATA FIGURE3.-Nucleotide sequence of the DBF4 gene. The translation is shown in the single-letter code below the nucleotide sequence. The numbers at the left count from the first base of the first ATG of the DBF4 ORF. The numbers at the right are the amino acid residue numbers of the ORF. The putative regulatory sequence ACGCGT at nucleotide -235 is underlined. strain KKY701 (dbf4::URA3/DBF4) was sporulated (ORF). It has been postulated that this and related and microdissected, and the spores were incubated at sequences regulate the expression of various DNA 24". In each tetrad dissected (total 24 tetrads ana- replication genes in a cell cycle-dependent manner lyzed), only two spores grew and bothwere Ura-. The (PIZZAGALLIet al. 1988). In fact, our previous studies inviable spores exhibited the same dumbbell shape as (CHAPMANand JOHNSTON 1989) showed that the lev- did dbf4-l and dbf4-2 cells incubated at the nonper- els of DBF4 transcript fluctuate during the cell cycle missive temperature. This result indicates that DBF4 with those of other DNA replication genes (JOHNSTON is essential for mitotic cell growth. et al. 1990). DBF4 nucleotide sequence: To obtain a knowledge of the DBF4 gene product, the nucleotide sequence DISCUSSION ofthe EcoRI-PstI fragmentderived from pKK616 The CDC7 gene encodes a protein kinase (PATTER- (Figure 1)was determined by the dideoxy-termination SON et al. 1986; BAHMANet al. 1988; HOLLINGSWORTH method. It revealed one large open reading frame and SCLAFANI1990) andits cell cycle execution point capable of encoding 695 amino acids with a predicted is justbefore S-phase. Beyond this point,protein molecular weight of 79,768 (Figure 3). Computer synthesis is no longer required for initiation of chro- analysis showed that DBF4 lacks the conserved amino mosome replication (HEREFORDand HARTWELL 1974; acid sequences found in protein kinases. However, PRINGLEand HARTWELL1981 ; CAMPBELL1986; using FASTA onthe UWGCG programrevealed NEWLON1988). It is, however, not known whether weak amino acid sequence similarity to theyeast SIR4 the CDC7 protein directly participates in the initiation gene (MARSHALLet aZ. 1987) (similarity 37.7%, iden- of chromosome replication. Moreover, the substrates tity 2 1.2%) andCAD protein (similarity 43%, identity for the CDC7 protein kinase are not known. In order 20.8%), although the biological significance of these to facilitate our understanding of the regulatory similarities is not yet clear. The predicted PI of the mechanism controlling the initiation of chromosome gene productis about 9.4; thus,it might bindto DNA. replication, we used a multicopy plasmid to isolate a The sequence -ACGCGT- (a MZuI restriction enzyme gene that suppresses the cdc7 mutation. This gene is recognition sequence) was found about 240 bp up- identical to DBF4, which we previously isolated by stream from the first ATG in the open reading frame complementation of dbf4-l. The dbf4 mutants were Suppressor of cdc7 Mutations 27 identified by theirdumbbell-shaped terminal mor- the dbf4 arrest,the executionpoint of DBFI was phology, which is characteristic of many DNA repli- mapped to a time in the cell cycle earlier than that of cation mutants, and by their defect in DNA synthesis CDC7 (JOHNSTON and THOMAS1982b). This result, at the restrictive temperature (JOHNSTON and THOMAS however, maynow beinterpretated in differently 1982a,b). Suppression of cdc7 by DBF4 is allele spe- from that offeredpreviously (JOHNSTONand THOMAS cific: a cdc7::LEU2 deletionmutation was not sup- 1982b): the amountof DBF4 protein is small, is likely pressed by DBF4, suggesting that DBF4 on a multicopy to be turned over rapidly during incubation at the plasmid is notable to bypass the CDC7 function. restrictive temperature and newly synthesized DBF4 Furthermore, this suppression is specific to mitotic protein is thus required for theprogression of the cell cells, because the sporulation defect of cdc7 mutants cycle after shift-down to the permissive temperature (SCHILDand BYERS1978) was not suppressed. Con- in dbf4 cells. Therefore, the levels of DBFI protein versely, CDC7 on a multicopy plasmid also suppressed may be of primary importance for theprogression of the temperature-sensitive dbf4-1, -2, -3 and -4 muta- the cell cyclethrough GI to S phase and may stimulate tions,but not the temperature-sensitive dbf4-5 and this progression by modulating the CDC7 protein ki- dbf4::URA3 deletion mutations. All temperature-sen- nase activity. BAHMANet al. (1988) have shown that sitive cdc7 mutations (cdc7-1, -2, -3 and -4) so far the CDC7 polypeptide made by in vitro translation has tested were incompatible with the temperature-sensi- a protein kinase activity. On the other hand, the other tive dbf4-1 and dbf4-2. These data suggest that the group has observed that the polypeptide expressed in CDC7 product directly interacts with andforms a E. coli does not have a protein kinase activity (YOON complex with the DBF4 product to facilitate initiation and CAMPBELL1991). Nonetheless, it will be interest- of chromosome replication. Because the DBF4 gene ing to learn whether the DBFI polypeptide further lacks amino acid sequence similarity to any known activates the CDC7 protein kinase made in vitro or in protein kinases (Figure 2), it is unlikely that it is E. coli. another protein kinase able to substitute for theCDC7 Other explanations for the suppression of cdc7 mu- function. Instead, like the CLNl, 2 and 3 gene func- tations by DBF4 are equally plausible. One is that tions which interact directly with the CDC28 protein overproduction of DBF4 protein nonspecifically sta- and activate the protein kinase activity (RICHARDSON bilizes the temperature-sensitive cdc7 protein, which et al. 1989; WITTENBERG,SUGIMOTO and REED 1990), thus becoming less temperature-sensitive. This, how- the DBFI protein may act as either an activator or a ever, is less likely because dbf4-l on a multicopy plas- modulator of the CDC7 product. However, other pos- mid failed to suppress cdc7 mutations (our unpub- sibilities are equally likely. One of these is that the lished results). Furthermore, a 4-bp insertion at the DBF4 gene productis a substrateof the CDC7 protein AccIII site ofDBF4 (nucleotide number 2375in Figure kinase. Another possibility is that the CDC7 and DBF4 3) generates a frameshift mutation producing a mu- proteins may act in a common biochemical pathway. tant gene (designated dbf4-6) encoding a polypeptide To distinguish between these possibilities, antibodies of 652amino acids (predicted molecular weight against either the CDC7 or DBF4 gene product may 74,780) lacking about 40 residues at the C terminus be useful. of the DBFI polypeptide. This mutant allele can com- Transcriptional regulationof CDC7 is very different plement all temperature-sensitive dbf4 mutations. fromthat of DBF4; the levels of DBF4 transcript However, it neithercomplemented the dbf4::URA3 fluctuate in the cell cycleand peak at the GI/S bound- mutation nor suppressed temperature-sensitive cdc7 ary (CHAPMANandJoHNsToN 1989), asdo otherDNA mutations (our unpublished results). This suggests replication-related gene transcripts (JOHNSTONet al. that the C-terminal regionof the DBFI polypeptide is 1990). On the other hand, thelevels of CDC7 mRNA essential for suppression of cdc7 mutations but is not are constant throughout thecell cycle (SCLAFANIet al. essential for DBF4 function when the full-size poly- 1988). Although not yet proven, it seems likely that peptide generated by temperature-sensitive dbf4 mu- the levels ofboth the CDC7 and DBFI proteins reflect tations is present. It also suggests that the DBF4 pro- the levels of their transcripts. If that is so, the CDC7 tein is multimeric inside the cell. product, which is synthesized throughoutthe cell dbf4 mutation was tentatively mapped at46 cM cycle, may be relatively stable and able to function for from the centromere on the left arm of chromosome several cell generations (SCLAFANIet al. 1988) before ZV (CHAPMANand JOHNSTON 1989). In that region of the protein is either diluted or inactivate. Onthe chromosome, another gene CDC2, which encodes a otherhand, the amount of DBF4 product is only catalytic subunit of DNA polymerase I11 (6), is located. enough for oneor two additional cell generations (this We have tried to map more precisely dbf4 mutation study). using cdc2 and rad55 mutations. However, we were Because new protein synthesis is still required be- not able to see any significant linkage between cdc2 fore initiation of DNA replication after release from and dbf4 [5 parental ditype (PD), 9 nonparental ditype 28 K. Kitada et al. (NPD) and 31 tetratypes (T) spores]. Contrary to the 1982 New temperature-sensitivemutants of Saccharomyces previous data (CHAPMANand JOHNSTON1989),we ob- cerevisiae affectingDNA replication. Mol. Gen.Genet. 187: 42-46. served a significant linkage between rad55 and dbf4 DYKSTRA,C. C., K. KITADA, A. B. CLARK,R. K. HAMATAKE and (35 PD, 2 NPD and 25 T) (K. KITADA and A. SUGINO, A. SUGINO, 1991 Cloning and characterization of DST2, the unpublished results). Therefore, it is likely that DBF4 gene for DNA strand transfer protein /3 from Saccharomyces is located on the right armof chromosome ZV, instead cerevisiae. Mol. Cell. Biol. 11: 2583-2592. of the left arm. This result may explain why two wild- GIETZ,R. D., andA. SUGINO,1988 New yeast-Escherichia coli shuttlevectors constructed with invitro mutagenized yeast type spores and two double-mutant spores (cdc7 dbf4) genes lacking six-base pair restriction sites. Gene74: 527-534. were unexpectedly predominant in the cross between HADWIGER, J. A.,C. WITTENBERG,M. D. MENDENHALLand S. I. cdc7 and dbf4 mutations, when we studied the incom- REED, 1989 The Saccharomyces cerevisiae CKSI gene, a hom- patibility of cdc7 and dbf4 mutations. olog of the Schirosaccharomyces pombe sucl+ gene, encodes a In the past, attempts have been made to isolate subunit of the Cdc28 protein kinase complex. Mol. Cell. Biol. 9: 2034-2041. extragenic suppressors of cdc7 mutations after muta- HASEGAWA,H., A. SAKAI and A. SUGINO,1989 Isolation, DNA genesis of cdc7 cells (our unpublished results). So far, sequence and regulation of anew cell division cycle gene from no dbf4 mutation has been identified, although other the yeast Saccharomyces cerevisiae Yeast 5: 509-524. mutations have been obtained (our unpublished re- HAYLES,J., D. BEACH, B. DURKACZand P. NURSE,1986 The sults). This might be due to incompatibility of dbf4 fission yeast cell cycle control genecdc2: isolation of a sequence sucl that suppresses cdc2 mutant function. Mol. Gen. Genet. with cdc7 mutations. 202: 291-293. As previously demonstrated (HAYLESet al. 1986; HEREFORD,L. M., and L. H. HARTWELL,1974 Sequential gene HADWICERet al. 1989),the approach used in this function in the initiation of S. cerevisiae DNA synthesis.J. Mol. study is powerful and may be used to identify new Biol. 84 445-461. genes which interact with any particular gene. More HOLLINGSWORTH,R. E., JR.,and R.A. SCLAFANI, 1990 DNA metabolism gene CDC7 from yeast encodes a serine (threonine) importantly, this is the first case providing genetical protein kinase. Proc. Natl. Acad. Sci. USA 87: 6272-6276. evidence of a possible direct interaction between the ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, CDC7 and other known cell cycle gene products. It 1983 Transformation of intact yeast cells treated with alkali also provides a clue to understanding how initiation cations. J. Bacteriol. 153: 163-168. of chromosome replication is regulated during thecell JOHNSTON, L. H., and A. P. THOMAS,1982a The isolation of new DNA synthesis mutantsin the yeast Sacchromyces cerevisiae. Mol. cycle. We hope to identify other genes whose proteins Gen. Genet. 186: 439-444. directly interact with the DBF4 polypeptide, using the JOHNSTON,L. H., andA. P. THOMAS, 1982b A furthertwo temperature-sensitive dbf4 mutations. mutantsdefective in initiation of the S phase in the yeast After this manuscript had been submitted,we were Saccharomyces cerevisiae. Mol. Gen. Genet. 186: 445-448. informed that DBF4 is allelic to DNA52 whose muta- JOHNSTON, L. H., J. H. M. WHITE, A. L. JOHNSON, G. LUCCHINI and P. PLEVANI, 1990 Expression of the yeast DNA primase tions were isolated by other group(DUMAS et al. 1982) gene, PRIl, is regulated within the mitotic cell cycle and in by sequence comparison and the cell cycle phenotype meiosis. Mol. Gen. Genet. 221: 44-48. in dna52-I was the same as in dbf4-1 (SOLOMONet al. LISZIEWICZ,J., A. GODANY, D. AGOSTONand H. 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