YEAST

VOL. 8: 273-289

(1992)

Genetic and Molecular Analysis of DNA43 and DNA52: Two New Cell-Cycle Genes in Saccharomyces cerevisiae NATALIE A. SOLOMON, MATTHEW B. WRIGHT, SO0 CHANG, ANN M. BUCKLEY, LAWRENCE B. DUMAS AND RICHARD F. GABER* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, U.S.A. Received 23 October 1991; accepted 3 December 1991

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna.52-1 cells undergo cell-cycle arrest at the restrictive temperature (37"C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna.52-I cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34. KEYWORDS - Saccharomyces

cerevisiae; cell-cycle genes; DNA synthesis.

INTRODUCTION

not been able to address adequately such issues as initiation, termination and regulation of DNA In vitro studies of DNA replication in highly diver- synthesis. gent organisms such as human, Drosophila and the We have pursued a genetic approach to identify yeasts have revealed that the enzyme complexes that additional factors that may be required for the mediate this function are remarkably similar. In proper control of DNA replication to gain insights each case a single complex consisting of a 125- to that complement those gained from in vitro studies. 180-kDa polypeptide containing DNA polymerase The analysis of conditionally lethal mutants of S . activity, two polypeptides of 50 and 60 kDa con- cerevisiae that are defective in S phase or the entry taining DNA primase activity and a 70- to 90-kDa into S phase has already led to the identification polypeptide of unknown function has been ident- of some of the genes involved in DNA replication. ified (Kaguni et af.,1983; Plevani et al., 1984; Wang For example, CDC17 encodes DNA polymerase I et al., 1984; Plevani et al., 1985). Structural aspects (Carson, 1987), CDC9 encodes DNA ligase (Barker of individual components of the complex are also and Johnston, 1983; Barker et al., 1985) and conserved. For example, human DNA polymerase CDC8 and CDC21 encode thymidylate kinase and a and Saccharomyces cerevisiae DNA polymerase I thymidylate synthetase, respectively (Bisson and share 31 % sequence similarity (Miller et al., 1988). Thorner, 1977; Jong et al., 1984; Sclafani and The DNA replication complex from S. cerevisiae Fangman, 1984), CDC7 encodes a kinase that can has been purified and its function studied in vitro phosphorylate histone H1 in vitro (Yoon and (Singh and Dumas, 1984; Singh et al., 1986; Pausch Campbell, 1990), and CDC2 encodes the large subet al., 1988; Brooks and Dumas, 1989; Brooke and unit of DNA polymerase I11 (Boulet et al., 1989; Dumas, 1991; Brooke et af.,1991).In vitro studies of Sitney et al., 1989). DNA replication, including these experiments, have In this report, we show that conditionally lethal *To whom correspondence should be addressed. mutations in two previously identified genes, 0749-S03X/92/O40273-17 $08.50 0 1992 by John Wiley & Sons Ltd

274

N. A. SOLOMON E r AL.

Table 1.

Strains used

Strain E. coliHBlO1 S . cerevisiae JL8 JL383 NSY-D43

NSY-D52 A364A-D5 NSY-D I4 NSY-D7 NSY-DI 7 NSY-a43 NSY-I0 MGGI5 R757 RI 174 M16-1 M17-1 11 1 4 c LDY14 146-4a 146-4b LDY298 LDY299 yMWl yMW2

Genotype

Source (reference)

hsdS2O recAI3 ara-I4proA2 lacYIgalK2 rpsL20 xyl-I5 4 - 1 4 supE44

Maniatis et al. (1982)

MATa dna43-I adeI ade2 ural tyrl his7lys2galI MATa dna52-I adel ade2 ural tyrl his7 lys2galI M A Ta/MATa dna43- I / dna43-I ura3-52/ura3-52 canI / ilv3/ M A Ta/MATa dna52- I ldna.52-I ura3-52/ura3-52 can I /canI MATaIMATa adelladel urallural tyrlltyrl his7/his7 lys2/ + l e d + galI/galI MATaIMATa cdcI4-IlcdcI4-I adeI/+ ade2/+ gall/+ his7/his71ys2/+ tyrI/+ urallural MATaIMATa cdc7-4/cdc7-4 adell + ade2/+ gall/ + his7/his7 l y s 2 / t tyrI/ + uraI/uraI M A T a / M ATa cdcI7-I/cdcI 7-I his7/his7 urallural MATa dna43-I ura3-52 leu2-3 MATa ura3-52 leu2-I,-112 ilv3 his3AcanI-II MATa cdc34 ura3-52 his3A M A Ta his4-I5 ura3-52 lys9 MATa trpIAI ura3-52 trkIA M A T a dna52::URA3 his4-IS ura3-52 lys9 MATa dna52::URA3 ura3-52 trpIAI trklA MATa his5 lysl I MATa adel ural his5 1ysII leu2 MATa his3A200 trpIAI trkIA2 ura3-52 M A T a trpIAI trkIA2 ura3-52 M A Ta dna52-I ura3-52 canI - I I M A Ta dna.52-I ura3-2 c a d - I I R757/R1174 146-4a/146-4b

Dumas et al. (1 982) Dumas et al. (1 982)

+

+

DNA43 and DNA52 (Dumas et al., 1982), confer a cell-cycle defect consistent with an essential role in the entry into or completion of S phase DNA replication. Upon shifting to a restrictive temperature, cells harboring dna43-I or dna52-I alleles arrest with a single large-bud and fail to undergo DNA replication. DNA sequence analysis of DNA43 and DNA52 indicates that they encode proteins of 59.6 kDa and 80.6 kDa, respectively. Gene disruption experiments confirmed that both DNA43 and DNA52 are essential for viability and genetic mapping experiments show that these genes reside at previously unidentified loci.

MATERIALS AND METHODS Strains and media Escherichia coli strain HBlOl (Maniatis et al.,

1982) was used to propagate all plasmids employed

M. Goebl Caber et al. (1988) Vidal et al. (1990)

in these experiments. The S. cerevisiae strains used in these studies are listed in Table 1. YPD medium consists of 1% yeast extract, 2% peptone and 2% dextrose. YN-5 contains 0.1 % yeast extract, 0.2% peptone, 50 mwsuccinic acid, 1 YOglucose, adjusted to pH 5.6. Following sterilization, 10% yeast nitrogen base (Difco) is added to a final concentration of @7%,and uracil and adenine are each added to a final concentration of 10mg/ml. YN-1 is made exactly as YN-5 except that uracil is added to a final concentration of 2 mg/ml. Quantitation of D N A synthesis in synchronous cell populations

Synchronous cell populations were derived from cultures by the method of Shulman and Hartwell (1973). Diploid S. cerevisiae strains were grown

275

GENETIC AND MOLECULAR ANALYSIS

to 3-6x lo6 cells/ml in I-liter cultures of YN-5 medium at 23°C. Cells were harvested, resuspended in 108 ml of 10% YN-I, 2.5% Dextran T40,20 mMglutathione, 10.5% Ludox HS-40 (Ludox HS-40 is a 40% colloidal silica suspension, the generous gift of E.I. duPont de Nemours & Co.) and divided into eight tubes. Density gradients were generated in a Sorvall SA-600 rotor, at 40,000 x g , for 30 min at 4°C. The gradients contained one large opaque band of cells near the midpoint with several minor bands above it. The cells in the minor bands were harvested with a pasteur pipet, filtered onto a nitrocellulose membrane (0.45 pm pore diameter) and suspended in YN-1 to a density of 5 x lo6 cells/ml. This suspension was divided into five 1.5-mlvolumes for morphological examination and five 4.5-ml volumes for DNA synthesis measurements. DNA synthesis was measured by adding [3H]uracilto a final concentration of 20 or 40 pCi/ml to the 4.5-ml samples. Identical cultures were incubated at the permissive (23°C) and restrictive (35°C or 37°C) temperatures while shaking in water baths. At appropriate times the 23°C cultures were shifted to the restrictive temperature (35°C or 37°C) for the remainder of the experiment. The mutants in the collection we screened were originally isolated by their inability to grow at 37°C. Since some of the non-congenic strains used for comparison in this study were unable to undergo normal cytokinesis at 37"C, the highest temperature (up to 37°C) that allowed normal cytokinesis to occur was used as the elevated temperature for these strains. Thus, wildtype (A364A-D5), cdcl4-1 and cdc7-4 strains were shifted to 35°C while the remaining strains were shifted to 37°C. At 20-min intervals throughout each 6-h experiment, 200 p1 samples were taken, added to 2 ml of I N NaOH, quick frozen and stored at -20°C. At the conclusion of the experiment all samples were thawed and incubated at 37°C for 18 h to hydrolyse RNA. DNA was then precipitated with trichloroacetic acid and collected on filters. Radioactivity incorporated into the DNA was quantitated by liquid scintillation. Rates of DNA synthesis were determined from slopes calculated from the approximately linear portions of the first S phase using the least-squares method. From two to six assays were performed for each strain and the ratios of the slopes at the restrictive vs permissive temperature were calculated. Cells were fixed for morphological examination by adding 50-pl samples taken from the 1-5-ml cultures to 250p1 of 7.4% formaldehyde. The formaldehyde-fixed cell samples were sonicated for

30 s and the fraction of single cells, small-budded cells and large-budded cells present was determined using phase contrast microscopy. Quantitation of R N A andprotein synthesis in synchronous cell populations

The measurement of RNA and protein synthesis was performed similarly to that for DNA synthesis. When measuring RNA synthesis the concentration of [3H]uracil was IOpCi/ml and when measuring protein synthesis [14C]aminoacids were added to a final concentration of 1 pCi/ml. 200-pi aiiquots of the cell cultures were added to trichloracetic acid and stored at -20°C until filtration. Radioactivity was measured using Beckman model LS 7000 scintillation counter. Cell morphology was examined as described above. Nuclear staining

Synchronous cell populations were prepared as above and divided for incubation at both permissive and restrictive temperatures. Morphology of the cells was monitored at 1 ,2 and 4 h of incubation at the restrictive temperature. Nuclear staining was performed in the following manner. The cells were pelleted by centrifugation, incubated for 30 min at room temperature in 1 ml 70% ethanol, pelleted again and incubated for 15min at room temperature in 1 ml of 0.1 pg/ml 4,6-diamidino-2-phenyl indole (DAPI) obtained from Sigma. The cells were then washed twice and resuspended in a final volume of 1 ml of distilled water, 200 p1 of each sample was centrifuged onto a slide and an anti-bleaching compound (0.1% p-phenylenediamine, 25 mMTri-HC1, pH 8.0, 75% glycerol) was added before each sample was sealed under a coverslip. Nuclear staining was observed using a Zeiss Axiophot photomicroscope. Plasmids

Plasmid vectors used in this study were YIp5 (Struhl et al., 1979), YCp50 (Rose et al., 1988), YEp24 (Botstein et al., 1979), pGEM4Z, pRG411, pRG415, pRS306 and pRS316 (Hinnebusch, 1988). pRG411 contains a 1.8 kb ClaI-BalI fragment of M13mp18 (Norrander et al., 1983) containing the polycloning and lacZ' regions inserted into the Clal and NruI sites of YIp5. pRG415 contains the M13amp18 fragment inserted into YCp50. Plasmid pMW2, containing a BamHI fragment that encompasses most of the DNA43 gene, was

276 generated by subcloning the 1.5-kbBamHI fragment from pNSl into the BamHl site of plasmid pJA5, a derivative of YCp50 missing the HindIII, EcoRI and ClaI restriction sites. A I.l-kb HindIII fragment containing the URA3 gene was then subloned from YEp24 into the unique HindIII site ofpMW2, generating plasmid pMW3. pMW3 thus contains a 2.6-kb BamHI fragment that consists of the URA3 gene flanked by DNA43 sequences (Figure 4). Disruption of DNA43 was performed by transforming a homozygous ura3-52/ura3-52 DNA43lDNA43 recipient (yMW 1) to uracil prototrophy with the gel-purified 2.6-kb BamHI fragment from pMW3. Plasmid pMW14 contains a 2.4-kb XbaI-EcoRI fragment that contains the entire DNA52 gene. A 1.5-kb BgnI fragment containing the TRPl gene was subcloned into the BgnI site of plasmid pMW14 to create plasmid pMW 15. The BglII site was shown by DNA sequence analysis to lie within the coding region of DNA52. The 3.9-kb XbaI-EcoRI fragment of plasmid pMW 15 thus contains the DNA52 gene disrupted by insertion of TRPl. This 3.9-kb fragment was gel purified and used to transform a homozygous trplAlltrp1A 1 DNA521DNA52 recipient (yMW2) to disrupt one copy of the gene. Yeast transformation Transformation of S. cerevisiae was performed by the cation method (Ito et al., 1983) using LiAc. DNA sequence analysis Restriction endonuclease fragments encompassing the DNA43 gene were subcloned into pGEM42. The DNA sequences of both strands of each fragment were determined by the dideoxy-chaintermination method of Sanger et al. (1977) performed on the double-stranded DNA templates following alkali denaturation (Toneguzzo et al., 1988). DNA sequences that spanned the junctions of the subclones were determined from a plasmid containing the entire DNA43 gene (pNS-6) using specific oligonucleotide primers chosen to hybridize near the junctions. The reagents used during the polymerization reactions, including SequenaseTM were obtained from United States Biochemical Corp. [35S]dATP(lOOCr1500 Ci/mol) was added to the polymerization reactions and the products were separated on 6% polyacrylamide (premixed gel solution from BRL) wedge gels (04-1.2 mm). The 2.4-kb EcoRI-XbaI fragment containing the DNA52 gene was subcloned into both pGEM4Z

N. A. SOLOMON ET AL.

and pRS316, creating plasmids pMW14 and pMWl7, respectively (Figure 4). A unidirectional set of nested deletions into the EcoRI end of the fragment was created by exonuclease 111digestion of EcoRI, KpnI-digested pMWl7 using the Pharmacia reagents for creating double-stranded nested deletions according to the directions provided. To sequence the opposite strand, another set of nested deletions was made into the XbaI end ofthe fragment by exonuclease I11 digestion of XbaI, SphI-digested pMW14. The DNA sequences of the two sets of nested deletions were determined as described above. Several oligonucleotide primers were used to determine the sequences of junctions between adjacent non-overlapping deletions using plasmid pMW14 as template for the reactions. DNA Manipulations Rapid plasmid DNA isolation was done by the method of Holmes and Quigley (1981). Restriction enzymes were from Bethesda Research Laboratories and were used according to the manufacturer’s directions. Restriction analysis, gel electrophoresis, and Southern analysis were performed as described by Maniatis et al. (1982). A DNA43-specific probe was prepared by gel purification of the 1.5-kb BamHI fragment from plasmid pNS-1 followed by 32P radiolabeling by the random primer method using oligolabeling reagents obtained from Pharmacia. A DNA52-specific probe was prepared in the same way after gel purification of the 2.6-kb XbaI-EcoRI fragment of plasmid pMW14. Filters containing the appropriate probe hybridized to genomic DNA were washed three times in 6 x SSC (1 x ssc is 0.15 M-NaCl plus 0.015 M-sodium citrate)-O.l% SDS at 55°C prior to autoradiography. Genetic mapping Standard linkage values were derived from tetrad data by using the equation X (in centiMorgans, cM) = 50 tetratype asci + 6 (non-parental ditype asci)]/total asci (Perkins, 1949). Gene order in multipoint crosses was determined by analysing recombinant asci containing cross-overs in the regions of interest. RESULTS dna43-1 and dna52-1 mutations confer defects in DNA synthesis dna 43-1 and dna52-1 mutations in strains JL8 and JL383 (Table 1) were previously shown to confer a

277

GENETIC AND MOLECULAR ANALYSIS

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30 +

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O

0

60

120

180

240L

Time (min) Figure I . RNA and protein synthesis measurements. Strains: wild-type (A364A-D5), dna43-I (NSY-D43) and dna52-1(NSY-D52). RNA and protein synthesis were monitored as described in Materials and Methods. Permissive temperature (0,23"C); restrictive temperature ( A , 35°C for wild-type, 37°C for dna43-I and dna52-I);(see text for details).

heat-dependent defect in DNA synthesis (Dumas et al., 1982). The effects of these mutations on macromolecular synthesis were further assessed by measuring the rates of DNA, RNA and protein synthesis in synchronous populations. The rates of macromolecular synthesis in synchronous cell populations of dna43-I and dna52-Icells that were shifted to the restrictive temperature were compared to cells that remained at the permissive temperature. The results of these experiments indicated that the rates of RNA and protein synthesis in these mutants are not significantly different from wild-type cells at either the permissive or restrictive temperatures (Figure 1). The point in the cell cycle at which DNA synthesis is affected in dna43-I and dna52-I mutants was determined by analysing DNA synthesis and cell morphologies in synchronous populations of wildtype and dna43-Ildna43-I or dna52-Ildna52-1 diploid cells. Synchronous cell populations were obtained by isopycnic density centrifugation

(Shulman and Hartwell, 1973) which yielded subpopulations that were highly enriched for precytokinesis cells (65-85% had large buds; see Materials and Methods). The synchronous populations were divided and portions were incubated at permissive (23°C) or restrictive (35°C or 37°C) temperatures. The morphology of these cells and their ability to synthesize DNA were determined from the analysis of samples taken at 20-min intervals. Morphology profiles of the cell populations were generated by determining the percentage of single cells, small-budded cells and large-budded cells in each sample. DNA synthesis profiles were generated by measuring the rate of [3H]uracil incorporation into alkali-resistant, acid-precipitable material. When incubated at the permissive temperature, both wild-type and mutant cell populations exhibited two discrete periods of DNA synthesis, corresponding to the S phases of two subsequent cell cycles (Figure 2). The rates of DNA synthesis at the

278 permissive temperature, determined by measuring the slopes of the curves, were similar in wild-type and mutant cells. In contrast, when incubated at the restrictive temperature not only did the mutant cells show significantly reduced rates of DNA synthesis during the first S phase but they failed to enter the second S phase (Figure 2). The ratio of the rate of DNA synthesis during the first S phase at the elevated temperature to the rate at the permissive temperature was 1.2 for the wild-type population; this ratio decreased to 0.5 for the dna43-I and dna521 populations. The residual DNA synthesis in the mutant cells at the restrictive temperature could have been due to a combination of mitochondria1 DNA synthesis, synthesis at specific sites of the genome such as telomeres, or leakiness of the mutant alleles. Cell morphology profiles showed that dna43-I and dna52-I cells progress through the cell cycle normally at the permissive temperature (Figure 2). In contrast, the profiles of the populations incubated at the restrictive temperature indicated a marked difference in the ability ofwild-type and mutant cells to progress through the next cell cycle. Although initially both wild-type and mutant cells proceeded into the next cell cycle at indistinguishable rates, the dna43-I and dna52-1 populations arrested as single cells containing a large bud (Figure 2). We tested the possibility that the defect in DNA synthesis of dna43-1 and dna52-1 cells might be a secondary effect due to an inability to complete the second cytokinesis. The rates of DNA synthesis in dna43-I and dna52-I cells were compared to that observed in cells containing a cdcl4 mutation, a conditional mutation known to confer defective cytokinesis at the restrictive temperature (Culotti and Hartwell, 1971). In contrast to dna43-I and dna52-1 cells, synchronous populations of wild-type (A364A-D5) and cdcl4 cells (NSY-D 14) exhibited increased rates of DNA synthesis at the elevated temperature compared to the permissive temperature (Fig. 2). Since the rate of DNA synthesis in cdcl4 cells during the first S phase at the restrictive temperature was twice that observed at the permissive temperature, the inability to undergo the second cytokinesis does not interfere with DNA synthesis. We next compared the DNA synthesis profiles of dna43-1 and dna52-I synchronous populations to that generatedfromapopulationofcellsknownto be defective in DNA polymerization, cells containing a conditional mutation in the gene encoding DNA polymerase I (cdcl7-1).As in dna43-I and dna52-1 cells, the rate of DNA synthesis in cdc-17-llcdcl7-1

N. A. SOLOMON ET AL.

diploidcells was significantly reduced (to 30%)at the restrictive temperature compared to the permissive temperature (Figure 2). This result further supports the likelihood that DNA43 and DNA52 encode products essential for DNA synthesis. The inability of dna43-I and dna52-I cells to undergo a second round of DNA synthesis following the shift to restrictive temperature combined with their terminal phenotype at growth arrest suggested that these mutants are defective either in S phase DNA synthesis or at a step late in GI , which results in their inability to enter S phase. To try to distinguish between these alternatives, we compared temperature-shift DNA synthesis profiles of dna43-I and dna52-I cells with the profile generated by cdc7-4 cells; cdc7-4 confers a conditional cell-cycle defect late in G1 (Culotti and Hartwell, 1971). cdc7-4/ cdc7-4 diploid cells that were shifted to restrictive temperature exhibited a delay in the onset of S phase and a slight decrease in the rate of S phase DNA synthesis compared with that observed at permissive temperature (Figure 2). Although the reduction in S phase DNA synthesis following the temperature shift was smaller in cdc7-4 cells compared to dna43-1 and dna52-1 cells, we could not conclude that dna43-I and dna52-I cells are defective in late GI as opposed to S phase. The nuclei of dna43-1 and dna52-1 cells arrest in an undivided state

Nuclear morphologies of dna43-1 and dna52-I cells arrested at the restrictive temperature were compared to those of wild-type, cdcl4-I, cdcl7-1 and cdc7-4 cells. Nuclei were examined by DAPIstaining 4 h after a shift to restrictive temperature. As expected, nuclei present at all stages of division were observed in wild-type cells. In contrast, dna43-I and dna52-I cells contained a single nucleus that had migrated through the isthmus of the motherdaughter cell neck and had arrested in an undivided state (Figure 3), a morphology identical to that observed in cdcl7-1 and cdc7-4 mutants (Culotti and Hartwell, 1971; Hartwell, 1973; Hartwell et al., 1973) but significantly different from that observed in the S phase-competent, nuclear divisiondefective cdcl4-1 mutants. The nuclei in cdcl4-1 mutant cells initiate division and arrest with most DAPI-stained material at the poles of the doublet cell (Culotti and Hartwell, 1971). These results further support the hypothesis that DNA43 and DNA52 play essential roles in S phase or at the G I /S phase boundary.

dna43-1

Time (min)

dna52-1

cdcl4-1

cdcl7-1

cdc 7-4

Figure 2. DNA synthesis and morphology measurements for wild type, dna43-I and dna52-I cell cultures. Strains: wild type (A364A-D5),dna43-1 (NSY-D43), dna52-1 (NSYD52), cdcll-1 (NSY-D14),cdcl7-1 (NSY-DI 7) and cdc7-4 (NSY-D7). DNA synthesis and morphology were monitored as described in Materials and Methods. (A) DNA synthesis panels: cultures grown at permissive temperature (23"C,0);restrictive temperature (35°Cor 3 7 T , A); shifted (at arrow) from permissive to restrictive temperature (A); see text for details (B) Morphology panels of cultures grown at permissive temperature (23°C).(C) Morphology panels of cultures grown at permissive temperature and shifted (see arrow in panel A) to the appropriate restrictive temperature. Morphology key: large-budded cells (0);single cells (+); cells with small bud (A).

wild-type

280

N. A. SOLOMON ETAL.

wild-type

dna43- I

dna52-I

Figure 3. Nuclear morphology in ha43-I and dna52-1 arrested cells. Synchronous populations of (A) wild-type (strain A364A-D5), (B) dna43-I(strain NSY-D43) and (C) dna52-1 (strain NSY-D52) cells were incubated at 37°Cfor 4 h, fixed and stained with the DNAspecific fluorescent dye, DAPI, as described in Materials and Methods.

Cloning of DNA43 and DNA52 DNA43 was cloned from a S. cerevisiae genomic library (Rose et al. 1988) by its ability to suppress the heat-sensitive growth defect in dna43-1 recipient cells. Six Ura' temperature-independent (Ts') transformants were identified among approximately 17,000 Ura' transformants obtained. The plasmids contained in the Ura' Ts' transformants were recovered and propagated by transformation of E. coli strain HBlOl. Restriction endonuclease digestion indicated that they represent four different plasmids containing overlapping S. cerevisiae DNA fragments that span a region of 20 kb. A partial restriction site map of the region of overlap is presented in Figure 4. A plasmid integration experiment was performed to show that the cloned insert contained the DNA43 gene. A 5.2-kb Xbal-Clal fragment encompassing the overlap region was subcloned into the integrative plasmid pRG411. The resulting plasmid, designated pNS-5.2 was linearized by cleavage at the two Hind111 sites within the cloned insert, and then used to transform a ura3-52 DNA43 strain (NSY-10) to Ura'. One of the Ura' transformants was crossed with a ura3-52 dna43-I strain (NSY-a43) and tetrad analysis ofmeiotic progeny obtained from the result-

ing diploid revealed complete genetic linkage between the integrated plasmid sequences (URA3) and the DNA43 locus (1 8PD:OTT:ONPD). Integration of pNS5.2 at the DNA43 locus demonstrated that the cloned inserts contain the authentic DNA43 gene. Transformation of the replicative plasmid pNS-6, containing a 3.1-kb Xbal-EcoRI fragment (Figure 4) into a dna43-1 ura3-52 recipient demonstrated that this subcloned fragment could suppress the temperature-sensitive phenotype. Based on the inability of other subclones to complement the dna43-I mutation, the functional DNA43 gene was likely to contain the Sac1 site and at least one of the BamHI sites indicated in Figure 4. DNA52 was cloned from the same S. cerevisiae genomic library by its ability to suppress the heatsensitive growth defect in dna52-I recipient cells. Six Ura' Ts' transformants were identified and the plasmids they contained were recovered and propagated by transformation of E. coli strain HBIOI. Restriction endonuclease digestion of these plasmids indicated that they contain different overlapping S. cerevisiae DNA fragments spanning a 20-kb region (Figure 4). A plasmid integration experiment was performed to show that the cloned plasmids contained the DNA52 gene. Subcloning experiments indicated

28 1

GENETIC AND MOLECULAR ANALYSIS

YCp50

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YCp50 YCp50 YCp50 YCp50

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r pRS316

Figure 4. Composite maps of restriction endonuclease sites determined from S. cerevisiue genomic fragments containing DNA43 and DNA52. The dnu43::URA3 and dna52::TRPI disruption constructs within the coding region of each gene are shown. Construction of subclones is described in Materials and Methods.

that the 2.4-kb Xbal-EcoRI fragment contained within the replicative plasmid pMW16 could suppress the temperature-sensitive growth defect of dna52-I recipient cells. This fragment was subcloned into the integrative plasmid pRS306 and the resulting plasmid, pMW 19, was linearized by cleavage at the single BgZII site within the cloned

insert, and then used to transform ura3-52 DNA52 strains (R757 and R1174) to uracil prototrophy. Ura' transformants were crossed with ura3-52 dna52-I strains (LDY298 and LDY299). Tetrad analysis of meiotic progeny obtained from resulting diploids revealed complete genetic linkage between the Ura+ and Ts' phenotypes (15PD:OTT:ONPD).

282 -346 -258 -168

-78 13

N. A. SOLOMON ETAL. AAAAAGGTTAAAGAGAGTCCAGCAAATGATCAAGCTTCCA~GATGTAATATTAAACAATGTAATTATATAAATATGAAACATC TACATATTTTAAATGTCACTAATGTCATTACAGAGGACATAAAGTGATTTATGACACATCCGTACTAGTAGTTAAGTATGAACAAATTTT GGGTTTATTTGCCATTTTTTTTC~GTTTCTTGGATGCGC~ACCCACCTTTTCTAACACCACTAAGAAATATCAACTTTATAGGC CATCGAAGATAAAGGAACGTAAGTTTGTCAATTCAACCTCACATTTTCAACGCACATAAGCACTTGGTTCGTGGAGAAATGAATGATCCT M N D P CGTGAAATTTTAGCGGTTGATCCGTACAATAATATTACTTCTGATGAAGAGGATGAGCAAGCCATCGCGAGAGAACTTGAATTTATGGAA R E I L A V D P Y N N I T S D E E D E Q A I A R E L E F M E

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CGAAAGAGGCACGCCTTAGTGGAACGATTAAAAAGAAAGCAAGAATTTAAGAAACCCCAGGATCCTAATTTTGAAGCCATCGAGGTACCT R K R H A L V E R L K R K Q E F K K P Q D P N F E A I E V P

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S

T

T

T

Y

F

M

E

K

F

Q

N

A

K

K

N

E

313

GATAAACAAATTGCCAAGTTTGAAAGCATGATGAATGCAAGAGTACATACGTTCAGTACCGATGAGAAGAAATATGTGCCGATAATCACA D K Q I A K F E S M M N A R V H T F S T D E K K Y V P I I T

4 62

4 63

AACGAATTAGAAAGCTTTTCAAATCTTTGGGTTAAAAAGAGGTACATACCTGAAGATGACTTAAAACGGGCTTTGCATGAGATCAAAATC N E L E S F S N L W V K K R Y I P E D D L K R A L H E I K I

552

553

CTTCGGTTGGGCAAACTTTTTGCTAAAATTCGCCCACCTAAATTTCAAGAGCCTGAATACGCCAACTGGGCCACCGTAGGCCTCATTAGC 642 L R L G K L F A K I R P P K F Q E P E Y A N W A T V G L I S

64 3

CACAAATCGGACATCAAATTTACATCATCTGAAAAGCCAGTCAAATTCTTCATGTTCACCATAACGGACTTTCAGCATACACTAGATGTT 7 3 2

H

K

S

D

I

K

F

T

S

S

E

K

P

V

K

F

F

M

F

T

I

T

D

F

Q

H

T

L

D

V 822

133

TATATCTTCGGGAAAAAGGGTGTAGAAAGATATTATAATCTTCGCCTGGGTGATGTGATAGCAATATTAAACCCAGAAGTACTACCATGG Y I F G K K G V E R Y Y N L R L G D V I A I L N P E V L P W

023

AGACCCTCAGGGCGAGGAAATTTTATCAAATCCTTCAACCTTCGAATTAGTCATGACTTCAAATGTATCCTGGAGATAGGTTCAAGTAGA 912 R P S G R G N F I K S F N L R I S H D F K C I L E I G S S R

913

GATTTAGGTTGGTGTCCCATAGTGAATAAAAAGACTCACAAAAAATGTGGCTCTCCCATTAACATATCTCTTCATAAGTGTTGCGATTAC 1 0 0 2 D L G W C P I V N K K T H K K C G S P I N I S L H K C C D Y

1003

CATAGAGAAGTGCAATTTCGTGGAACAAGTGCTAAAAGAATTG~TTAAATGGTGGGTACGCCTTGGGCGCGCCTACGAAAGTGGACTCT H R E V Q F R G T S A K R I E L N G G Y A L G A P T K V D S

1092

1093

CAACCAAGCCTATATAAGGCCAAAGGGGAAAACGGGTTTAATATAATCAAAGGTACTCGTAAGCGCCTGTCAGAAGAGGAGGAAAGACTT Q P S L Y K A K G E N G F N I I K G T R K R L S E E E E R L

1182

1183

AAAAAGAGCTCTCACAATTTTACGAATAGTAATTCTGCCAAAGCATTTTTCGACGAGAAATTTCAGAATCCAGATATGCTGGCAAACTTA K K S S H N F T N S N S A K A F F D E K F Q N P D M L A N L

1272

1213

GACAATAAAAGAAGGAAAATAATAGAAACTAAGAAATCGACAGCACTGAGCCGCGAACTAGGCAAAATTATGAGAAGGAGGGAATCCAGC D N K R R K I I E T K K S T A L S R E L G K I M R R R E S S

1362

1363

GGATTAGAACATAAGAGCGTCGGAGAGCGACAGAAAATGAAACGAACCACAGAAAGTGCCCTCCAGACAGGGCTTATCCAACGCCTAGGA G L E H K S V G E R Q K M K R T T E S A L Q T G L I Q R L G

1452

1453

TTCGATCCCACTCATGGAAAAAATTTCCCCAAGTACTCAAGTCTTCTGTATCAGGGAGCGAACCTAAGAACAACTTACTCGGTAAAAAAA 1 5 4 2 F D P T H G K N F P K Y S S L L Y Q G A N L R T T Y S V K K

1543

AAACTGTTATAAACGACGATCTCTTGCATTACAAGAAGGAAAAAGTCATTCTCGCACCTTCAAAGAACGAATGGTTCAAGAAAAGAAGCC K

1633 7 723

L

L

1632

*

ATCGCGAAGAAGTTTGGCAAAAAACATTTCGGATCCAAGGAAACTAAAGAAACTTCTGACGGTAGTGCCAGCGATCTTGAGATAATATAA

ATAAGTATTGGTGTGCTTTTCAGGGCCCCAAAATG

1722

1757

Figure 5. NucleotidesequenceofDNA43.Thenucleotidesequenceofthere~onfromtheXbaI site to theEcoRIsiteinplasmidpNS-6 was determined as described in Materials and Methods. A 1551 bp open reading frame was identified. Sequences related to the ACGCGT motif present in the 5’ non-coding region of other S phase-specific genes are underlined.

283

GENETIC AND MOLECULAR ANALYSIS

Integration of pMW19 at the DNA52 locus demonstrated that the cloned insert contains the authentic DNA52 gene.

expression may be regulated by the same cell-cycle control mechanisms as the expression of other DNA-replication genes, supporting their possible role in DNA replication.

DNA sequence analysis of DNA43 and DNA52

The DNA sequence of the 3-1-kb Xbal-EcoRI fragment containing the functional DNA43 gene (Figure 5 ) was determined as described in Materials and Methods. The single long open reading frame of 1551 nucleotides found within this DNA fragment is capable of encoding a protein of 59.6 kDa. As predicted from the subcloning experiments, the DNA43 open reading frame contains the Sac1 site and one of the BamHI sites. A non-canonical TATA sequence (GATAAA) is present 71 nucleotides upstream of the first ATG of the open reading frame. Although a canonical TATAAA sequence is present further upstream (- 277), several ATG sequences are encountered prior to the first ATG of the large open reading frame. A sequence highly related to the consensus sequence determined to function in transcription termination, TAG. . . TAGT.. . (A-T rich). . .TTT (Zaret and Sherman, 1982) is present 125 nucleotides downstream of the TAA termination codon at the 3' end of the open reading frame. The sequence AAGAA is present six times between the 3' end of the open reading frame and the putative transcription termination signal but the significance of these sequences is unknown. The DNA sequence of the 2.4-kb Xbal-EcoRI fragment containing the functional DNA52 gene (Figure 6 ) was determined as described in Materials and Methods. One open reading frame of 21 12 nucleotides capable of encoding a protein of 80.6 kDa was identified in this fragment. A noncanonical TATA sequence (TAGAAA) is present 18 nucleotides upstream of the first ATG of the open reading frame. Since a DNA fragment, containing only 41 nucleotides upstream of the ATG start codon, is sufficient to suppress the temperaturesensitive phenotype of dna52-I recipient cells (Figure 4), the TAGAAA sequence may represent the authentic DNA52 promoter. The sequence ACGCGT, observed in the 5' non-coding region of a number of genes required for DNA replication and expressedjust prior to S phase (McIntosh et al., 1988; Pizzagalli et al., 1988) was found at position - 239. Two sequences closely related to the ACGCGT motif, ACGAGT and ACGCGG, were found upstream of DNA43 (at positions - 306 and - 145, respectively).The presence of these sequences in the DNA43 and DNA52 genes suggests that their

DNA43 and DNA52 are essential genes in S . cerevisiae To confirm that DNA43 is essential for viability, we constructed a disruption by insertion of URA3 within the coding region of the gene (see Materials and Methods) and used this construct to generate a null allele of DNA43 in vivo through a one-step gene-replacement experiment (Rothstein, 1983). The DNA fragment containing the dna43::URA3 disruption allele was used to transform a homozygous ura3-52/ura3-52 DNA43/DNA43 diploid strain (yMW1, Table 1) to a Ura' phenotype. Six Ura+transformantswere sporulated and a total of 24 asci were dissected by micro-manipulation onto rich medium. All of the dissected asci exhibited a 2 :Zsegregation pattern for viability. The two surviving spores from each tetrad were unable to grow on medium lacking uracil, indicating that the integrated URA3 gene had disrupted an essential gene. Southern analysis of BamHI-digested genomic DNA prepared from the pre-transformed diploid (yMWl), two Ura' transformants (yMW1-1 and yMW1-2), and two pairs of surviving Ura- meiotic progeny from the Ura' transformed diploids demonstrated that integration of the URA3containing fragment disrupted the DNA43 locus. Hybridization of a radiolabeled 1 -5-kb BamHI fragment, encompassing most of the DNA43 gene, to the genomic blots indicated that the pre-transformed diploid and all viable spores contain a single 1.5-kb DNA fragment, whereas the Ura' transformed diploid contains an additional 2 6 k b fragment homologous to the probe (Figure 7a). The size of this additional fragment is consistent with the insertion of the I.l-kb fragment containing the URA3 gene at the DNA43 locus. These results demonstrate that DNA43 is essential for viability in S. cerevisiae. In a similar manner, DNA52 was disrupted by insertion of T R P l within the coding region of the gene. This construct was used to generate a null allele of DNA52 by transformation of a trplAlltrplAl DNA521DNA.52 diploid strain (yMW2, Table 1) to a Trp+ phenotype (see Materials and Methods). Three Trp+ transformants were sporulated and a total of 30 asci were dissected by micro-manipulation onto rich medium. All of the dissected asci exhibited a

+

284

N. A. SOLOMON ETAL. -251 -221 -131 -41

TTTTTTTTGCAA&X€GXTTAGGTCGATC TTTGCGAAAAAGAAAAAAATAAAGAAATAACGATAATAATAACGGTAATGGCTCAGGCCAAGAAAAACAAAAATCAAGTTGCATAGTCTA TACTCAGCCAGGTGTAGATTCCTTTAGATCTGAATTCGCTTGCAGGGCTAGTTATCTTCAGAATTCAAGAAAGGTTTGAATCTGAAAGGCATTT GGAATTCTGCTTTTGGTTCATATTTAGAAAAGAAAGAAAGAAGAAAATGGTTTCTCCAACGAAAATGATAATAAGGTCTCCGTTAAAGGAAACA

M

V

S

P

T

K

M

I

I

R

S

P

L

K

E

-222 -132 -42 48

T

GATACCAACTTGAAACATAATAATGGAATTGCCGCATCAACAACAGCAGCGGGCCACTTGAATGTGTTTTCTAACGACAATAATTGCAAC D T N L K H N N G I A A S T T A A G H L N V F S N D N N C N

138

139

AATAACAATACCACTGAATCCTTTCCGAAGAAAAGATCTCTTGAGCGCCTCGAGCTCC~CAGCAGCAGCATTTGCATGAAAAGAAAAGAAAGG N N N T T E S F P K K R S L E R L E L Q Q Q Q H L H E K K R

228

229

GCCAGAATAGAAAGGGCCAGGTCTATTGAAGGCGCTGTCCAAGTCAGCAAAGGTACGGGTCTGAAAAATGTCGAGCCAAGGGTTACGCCT A R I E R A R S I E G A V Q V S K G T G L K N V E P R V T P

318

319

AAGGAATTGCTGGAATGGCAAACAAATTGGAAAAAGATAATGAAAAGAGATTCTCGCATTTACTTTGACATTACTGATGATGTAGAGATG K E L L E W Q T N W K K I M K R D S R I Y F D I T D D V E M

408

AATACATATAATAAGTCCAAGATGGACAAACGCAGAGATTTATTGAAAAGAGGGTTTCTTACATTGGGTGCGCAAATAACTCAATTTTTT

4 98

49

409

N

T

Y

N

K

S

K

M

D

K

R

R

D

L

L

K

R

G

F

L

T

L

G

A

Q

I

T

Q

F

F

499

GACACTACTGTCACAATAGTTATCACAAGAAGGTCTGTTGAGAACATATATTTACTAAAAGATACCGACATTTTATCGAGAGCTAAAAAA D T T V T I V I T R R S V E N I Y L L K D T D I L S R A K K

588

589

AACTACATGAAAGTTTGGAGTTACGAAAAGGCTGCCAGATTTCTGAAAAATCTTGATGTTGATTTGGATCATTTGAGCAAGACTAAATCT N Y M K V W S Y E K A A R F L K N L D V D L D H L S K T K S

678

679

GCTTCTTTAGCTGCGCCCACATTGTCCAATCTTCTACACAATGAAAAATTATATGGACCAACGGATAGAGACCCCAGAACTAAAAGAGAC A S L A A P T L S N L L H N E K L Y G P T D R D P R T K R D

768

769

GATATTCACTACTTTAAATATCCTCATGTATACCTTTATGACCTATGGCAAACTTGGGCCCCCATAATAACTTTGGAATGGAAACCTCAA D I H Y F K Y P H V Y L Y D L W Q T W A P I I T L E W K P Q

858

859

GAACTAACAAACTTAGACGAACTACCTTACCCCAATATTGAAAATAGGTTCATTCGGAAGATGCCCTTTTATAGGGGATAGGAATTATGAC

948

E

L

T

N

L

D

E

L

P

Y

P

I

L

K

I

G

S

F

G

R

C

P

F

I

G

D

R

N

Y

D

GAAAGTTCTTATAAGCGCGTAGTAAAGAGATACTCGAGAGACAAAGCAAACAAAAAATATGCACTGCAACTTCGTGCTCTATTTCAATAT E S S Y K R V V K R Y S R D K A N K K Y A L Q L R A L F Q Y

1038

1039

CATGCCGACACCTTACTGAATACGTCATCAGTTAATGATCAAACGAAAAACCTAATATTCATACCTCACACATGCAACGATTCTACCAAG H A D T L L N T S S V N D Q T K N L I F I P H T C N D S T K

1128

1129

AGCTTCAAAAAATGGATGCAAGAAAAGGCAAAAAATTTTGAGAAGACCGAGTTAAAGAAGACGGATGATAGCGCAGTTCAAGATGTTCGT S F K K W M Q E K A K N F E K T E L K K T D D S A V Q D V R

1218

1219

AATGAACATGCTGACCAAACCGATGAAAAAAATAGTATATTATTAAATGAAACTGAAACCAAAGAGCCTCCGTTGAAAGAAGAAAAAGAA N E H A D Q T D E K N S I L L N E T E T K E P P L K E E K E

1308

1309

AATAAACAATCTATAGCAGAAGAATCGAATAAGTACCCACAGCGAAAAGAGCTGGCTGCCACACCAAAACTAAACCATCCAGTATTAGCT N K Q S I A E E S N K Y P Q R K E L A A T P K L N H P V L A

1398

1399

ACTTTTGCAAGGCAAGAAACTGAAGAAGTGCCGGATGATTTGTGCACTTTGAAAACAAAGTCACGTCAGGCATTTGAAATCA~GCAAGT 1 4 8 8 T F A R Q E T E E V P D D L C T L K T K S R Q A F E I K A S

1489

GGTGCACATCAATCTAATGATGTGGCAACCTCTTTTGGCAATGGTTTGGGCCCAACAAGAGCAAGCGTCATGAGTAAGAACATGAAGTCA G A H Q S N D V A T S F G N G L G P T R A S V M S K N M K S

1578

1579

TTAAGTAGACTAATGGTTGATAGAAAGCTGGGAGTAAAGCAGACAAATGGAAATAACAAAAATTATACAGCCACTATAGCAACTACTGCT L S R L M V D R K L G V K Q T N G N N K N Y T A T I A T T A

1668

1659

GAAACATCAAAGGAAAATAGACACAGATTAGATTTTAATGCTTTGAAAAAAGACGAAGCCCCTTCGAAAGAGACGGGCAAAGATAGTGCT E T S K E N R H R L D F N A L K K D E A P S K E T G K D S A

1758

1759

GTACACTTAGAAACTAATAGAAAGCCCCAGAATTTCCCTAAGGTAGCTACCAAATCAGTCTCCGCAGACTCCAAAGTTCATAATGACATC V H L E T N R K P Q N F P K V A T K S V S A D S K V H N D I

1848

1849

AAGATAACAACCACAGAATCTCCAACAGCATCGAAGAAATCAACTTCCACAAACGTCACCTTACATTTTAACGCACAGACAGCACAGACA K I T T T E S P T A S K K S T S T N V T L H F N A Q T A Q T

1938

1939

GCACAGCCGGTGAAGAAAGAAACGGTAAAAAATTCCGGATACTGTGAAAATTGTCGTGTAAAATACGAATCTTTAGAACAACACATAGTT A Q P V K K E T V K N S G Y C E N C R V K Y E S L E Q H I V

2028

2029

TCTGAGAAGCATTTGTCTTTCGCTGAAAACGATTTAAATTTTGAGGCTATTGACTCGTTAATTGAAAATCTCAGATTTCAAATATAGGGA S E K H L S F A E N D L N F E A I D S L I E N L R F Q I *

2118

949

2119

CACGACGTAAAGTGCAGTAGCTTTTAGTGATAAAATCAAAATAGTATTGTTCCGTTTCCATTGCTGCTCGGAACAAAAAAGCTATCAACG

2208

,209

~CAATOTTATTGAATCACTTTCTCATTCACCCTT~~TACTTTCTTGCTATTGACTTAACTCTTATTTACTCGTCCATATATTATCAATTG

2298

2299

CATATATATACATATACATATATATATTATCATCTAGA

2336

GENETIC AND MOLECULAR ANALYSIS

285

2 + :2-segregation pattern for viability and the two surviving spores from each tetrad were unable to grow on medium lacking tryptophan. Southern analysis was performed with XbalEcoRI-digested genomic DNA from the pretransformed diploid (yMW2), two Trp' transformants (yMW2-I and yMW2-2), and two pairs of surviving Trp- meiotic progeny. Hybridization of a radiolabeled DNA52-specific probe to the genomic blot detected an additional fragment of 3.9 kb present in the Trp+ transformed diploid as well as the expected 2.4-kb fragment (Figure 7b). Only the 2.4-kb fragment was detected in genomic DNA of the pretransformed diploid and surviving Trp- progeny. The size of its additional fragment corresponds to insertion of the 1.5-kb fragment containing the T R P l gene at the DNA52 locus. These results demonstrate that DNA52 is also essential for viability in S. cerevisiae.

A single ascus was obtained that was tetratype for the his5-lysll marker pair and tetratype for the his5-dna43-I marker. This ascus was likely to have arisen due to a single crossover between lysl I and his5 and between his5 and dna43-I. From these data, the most likely gene order is CENZX-lysll-his5dna43-I (Figure 8a). Hybridization of a radiolabeled DNA fragment containing DNA52 to electrophoretically separated S. cerevisiae chromosomes immobilized on a filter (Carle and Olson, 1984, 1985) indicated that DNA52 is located on chromosome IV (data not shown). Analysis of meiotic progeny from a genetic cross between a DNA52:: URA3 trpl A1 strain M 17-1 (generated by transformation of strain RI 174 with the integrative plasmid pMW19) and a T R P l strain (R757) indicated that DNA52 lies on the right arm of this chromosome approximately 33.3 cM from T R P I . Precise mapping of the DNA52 locus was determined by crossing a DNA52:: URA3 strain (M17-1) with a cdc34 strain (MGG11). Tetrad Genetic mapping of DNA43 and DNA52 analysis of 163 asci (148 four-spored, 15 threeGenetic mapping of DNA43 revealed that it is spored) obtained from this cross confirmed that a newly identified locus. The location of DNA43 DNA52 is located 0-9 cM from cdc34 on chromowithin the S . cerevisiae genome was determined by some IV (Figure 8b). In addition, the genetic hybridizing a radiolabeled DNA fragment contain- distance between Trpl and DNA52 was refined to ing DNA43 to electrophoretically separated s. 30.4cM by analysis of this cross. cdc34-I and cerevisiae chromosomes (Carle and Olson, 1984, dna52-1 mutations complement each other (data 1985). The results of these experiments indicated not shown), indicating they are alleles of different that DNA43 is located on chromosome IX (data not genes. In addition, the DNA sequences of these shown). Genetic crosses between dna43-1 and other genes are unrelated (M. Goebl, personal communichromosome IX markers indicated that dna43-1 lies cation). The order of DNA52 with respect to cdc34 on the left arm of this chromosome. A three-point was not determined. These results indicate that cross was performed by mating a dna43-I strain DNA52 resides at a newly identified locus. (NSY - a43) to a his5 l y s l l strain (1 I14C). Tetrad analysis of 68 asci obtained from this cross demonstrated that dna43-I lies approximately 32 cM distal DISCUSSION from the his5 locus. Of 32 asci that were tetratype for In a search for genes involved in S phase DNA synthe his5-lysll marker pair, 18 were the result of thesis in S. cerevisiae, we screened a collection of multiple crossovers and were not considered for the conditionally lethal mutants by monitoring DNA determination of gene order. The remaining 14 asci synthesis in synchronous populations of cells isowere tetratype with respect to the lysll-dna43-I lated by isopycnic density centrifugation. Two marker pair. These asci were likely to have arisen mutants from this collection, dna43-I and dna52-1, from a single crossover between l y s l l and his5. exhibited decreased rates of DNA synthesis consistForty asci that were tetratype for the his5-dna43-1 ent with a specificdefect in S phase. Cells containing marker pair remained parental ditype for the his5- null alleles of DNA43 or DNA52 were inviable and lysl I marker pair. These asci probably arose as the germinating spores harboring such alleles underresult of a single crossover between his5 and dna43-I. went cell cycle arrest after forming a single large

Figure 6. Nucleotide sequence of DNA52. The nucleotide sequence of a DNA fragment from the XbaI site to the EcoRI site in plasmid pMW 16 was determined as described in Materials and Methods. A 21 12 bp open reading frame was identified. The ACGCGT motif found in the 5' non-coding region of other S phase-specificgenes is underlined.

286

N. A. SOLOMON ETAL.

bud. This terminal phenotype was identical to the morphology of dna43-1 and dna52-1 strains at cellcycle arrest when incubated at non-permissive temperature. Genetic mapping of DNA43 and DNA52 and their DNA sequences indicated that they have not been previously identified. DNA43 and DNA52 are required for the cell to enter or traverse S phase. Cells harboring the dna43-1 or dna52-1 mutations are markedly defective in DNA synthesis. When incubated at the restrictive temperature, dna43 and dna52 mutants exhibited h

.\$’

A

2.6

---+

1.5+

B 3.9 + 2.4 +

significantly decreased rates of DNA synthesis (2540% of the rate of DNA synthesis-competent cells). Several lines of evidence suggest that the lower rates of DNA synthesis in these mutants are due to specificeffects on DNA synthesis and are not simply secondary consequences of other cell-cycle defects. (i) The synthesis of RNA and protein occurs normally at both the permissive and restrictive temperatures, indicating that these mutations do not affect the synthesis of other macromolecules. (ii) cdcl4 cells, which fail to undergo cytokinesis at restrictive temperature, do not exhibit decreased rates of DNA synthesis under this condition. Therefore, the effects on DNA synthesis are not a consequence of an inability to complete cell division. (iii) Cells harboring a mutation in the gene which encodes a protein directly involved in DNA synthesis (cdcl7-1) have DNA synthesis profiles similar to dna43-1 and dna52-1 strains. The morphology of the nuclei at cell-cycle arrest can also help define the point at which the defect due to themutations occurs. Mutants that arecompetent for completion of S phase but defective at some point in M phase arrest with partially divided nuclei. Mutants defective in S phase, however, show undivided nuclei arrested at the isthmus of dividing cells. We monitored nuclear morphology at cellcycle arrest of a cdcl4-1 strain, which is competent for S phase DNA synthesis at the restrictive temperature. We observed that nuclear division at cell-cycle arrest is almost complete with most of the newly replicated DNA found at the poles of dividing cells. In contrast, the nuclei in cdcl7-1, cdc7-4, dna43-1 and dna52-1 strains at cell-cycle arrest

Figure 7. Southern blot hybridizationanalysis ofgenomic DNA containing wild-type, dna43:: URA3 and dna52:: TRPl alleles. (A) Lanes 1 and 2 contain genomic DNA from a DNA43/DNA43 diploid and a DNA43/dna43::URA3 diploid respectively. Lanes 3 through 6 contain genomic DNA of two pairs of viable sister spores obtained from the second diploid. An additional band corresponding to the dna43;:URAJ allele is present in the DNA43/dna43::URA3 diploid but not in any of its viable meiotic progeny. This indicates that disruption of DNA43 results in the inviability of the other two spores ofeach ascus. (B) Lanes 1 and 2 contain genomic DNA from a DNA52IDNA52 diploid and a DNA52/dna52::TRPI diploid, respectively. Lanes 3 through 6 contain genomic DNA of two pairs of viable sister spores obtained from the second diploid. All lanes contain a single band corresponding to the wild-type DNA52 allele, with an additional band corresponding to the dna52::TRPI allele present only in the DNA52/dna52::TRPI diploid but not in any of its viable meiotic progeny. This indicates that disruption of DNA52 also results in the inviability of the other two spores of each ascus. Details of these experiments are described in Materials and Methods.

287

GENETIC A N D MOLECULAR ANALYSIS

a) IX-L

b) IV-R

2cM

0.9cM

Figure 8. Genetic map positions of h a 4 3 and dna52. A left arm of chromosome IX depicting the position of dnaO with respect to other genetic markers (b) Right arm ofchromosome IV showing the location of dna52. The parentheses indicate that the orientation of dna52 with respect to cdc34 is undetermined. Genetic mapping experiments are described in the results section.

had migrated to the neck of the budded cell but remained undivided. This suggests that DNA replication has either not been completed or proceeds so slowly that cells lose nuclear division competence. Since cells harboring cdc7-4, a late G1 mutant, have the same morphology as dna43-I and dna52-1, it remains possible that DNA43 and DNA52 act just prior to initiation of DNA synthesis. The effects of dna43-1 and dna52-1 mutations on DNA synthesis and nuclear morphology indicate that these genes are required for essential functions in late G1 or early S phase. In an attempt to understand these functions, we cloned and analysed the DNA43 and DNA52 genes. Both genes were cloned from a S. cerevisiae genomic library by their ability to relieve the temperature-sensitive growth defects of the corresponding mutant alleles. DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. No significant similarities were found between the inferred DNA43 and DNA52 protein sequences and sequences contained in the available databases using the GCG program TFASTA. The sequence ACGCGT, which has been found in the 5‘ non-coding region of eight genes that are expressed just before S phase, is present in DNA52. DNA43 contains two sequences closely related to this motif. This observation further supports the hypothesis that DNA43 and DNA52 are S phase-specific genes. ACKNOWLEDGEMENTS N.A.S. and M.B.W. should be considered equal first authors. This work was supported by grants from

the National Institutes of Health (GM23443) to L.B.D. and the National Science Foundation (DCB-8657150) to R.F.G. Note Since we submitted our manuscript we became aware of a manuscript (Kitada, K., Johnston, L. H., Sugino, T. and Sugino, A.) describing DBF4, the DNA sequence of which appears to be essentially identical to DNA52. The authors report that overexpression of DBF4 can suppress the temperature sensitivity of several cdc7 alleles and, conversely, overexpression of CDC7 can suppress the temperature sensitivity of a dbf4 mutant. These results further support the likelihood that DNA52 plays a role in S phase or entry into S phase and suggest a direct interaction with the kinase encoded by CDC7. Nucleotide sequence accession number The nucleotide sequences reported here have been entered in the GenBank nucleotide sequence data library under accession numbers M83540 (for the DNA43-containing fragment) and M83539 (for the DNA52-containing fragment). REFERENCES Barker, D. G., Johnson, A. L. andJohnston, L. H. (1985). An improved assay for DNA ligase reveals temperature sensitive activity in cdc9 mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 200,458-462. Barker, D. G. and Johnston, L. H. (1983). Saccharomyces cerevisiae cdc9, a structural gene for yeast DNA ligase

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