Copyright 0 1993 by the Genetics Society of America

Conditional Mutations in the Yeast DNA Genes Affect Different Aspects of DNA Metabolism and Interactions in the DNA a-Primase Complex

Maria Pia Longhese,* Luca Jovine,* Paolo Plevani*and Giovanna Lucchini**+ *Dipartimento di Genetica e di Biologia dei Microrganismi, Uniuersita di Milano, 20133 Milano, Italy, and Tlstituto di Genetica, Universita di Sassari, 07100 Sassari, Italy Manuscript receivedJuly 28, 1992 Accepted for publication October2 1, 1992

ABSTRACT Different pril and pri2 conditional mutants of Saccharomyces cereuisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show anenhanced rate of bothmitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity oftheir defects in cell growth and DNA synthesis. These effects might be attributableto the formation of nicked and gapped DNA molecules thatare substrates for recombination anderror- prone repair, due to defective DNA replication in the primase mutants. Furthermore, pril and pri2 mutations inhibit sporulation and affect spore viability,with the unsporulated mutant cells arresting with a single nucleus, suggesting thatDNA primase playsa critical roleduring meiosis. The observation that all possible pairwise combinations of two pril and two pri2 alleles are lethal provides further evidence for direct interactionof the primase subunitsin vivo. Immunopurification and immunopre- cipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase a, and either mediatesor stabilizes association ofthe p48 polypeptidein the DNA polymerase a-primase complex.

N the yeast , as in all eukar- to use in vitro reconstitution studies tofurther analyze I yotic systems analyzed so far, a DNA primase their specific role in determining activity and activity is associated with DNA polymerase a (pola) their interactions. in a four-subunitcomplex that appearsto play a major To address these questions and togain some infor- role in initiation of DNA replication at an origin and mation about the function of DNA primase in differ- in priming and elongation of Okazaki fragments on ent aspects of DNA metabolism, we have produced the lagging strand (LEHMANand KAGUNI 1989; TSUR- conditional pril and pri2 mutants by random muta- IMOTO, MELENDY and STILLMAN1990). The yeast genesis of the cloned genes and replacement of the complex can be separated into two fractions, one of wild-type copy atthe correspondingchromosomal which, containing the p48 and p58 polypeptides, is locus (FRANCESCONIet al. 1991). Preliminary charac- able to synthesize short RNA primers (9-1 1 nucleo- terization of different recessive alleles, i.e., the leaky tides in length) that can be elongatedby pola (PLEVANI and temperature-sensitive (ts) pril-1, the cold-sensi- et al. 1985; BROOKSand DUMAS1989). Cloning and tive (cs) pril-2, the ts pri2-1 and the ts pri2-2 alleles, characterization of the PRIl and PRI2 genes encod- which all carry single base-pair substitutions causing a ing, respectively, the p48 and p58 protein species, change in amino acid residues conserved in the cor- have shown that they are both unique in the haploid respondingmouse polypeptides, has allowed us to yeast genome and essential for cell viability, and that assess the essential role of DNA primase in DNA the amino acid sequence of their encoded products is replication in vivo. In fact, the primary defect in all highly homologous tothat of thecorresponding these mutants, after shifting nonpermissiveto temper- mouse primase polypeptides (PLEVANI, FRANCESCONI ature, is an impairment in DNA synthesis, ranging and LUCCHINI1987; FOIANIet al. 198913; PRUSSAK, betweena complete arrest in the pri2-1 mutant to ALMAZANand TSENC1989; FRANCESCONIet al. 1991). only a doubling of the time needed to complete one Earlier studies with anti-p48 and anti-p58 antibodies round of DNA replication in the prz2-2 mutant. These and an affinity labeling procedure suggest that both defects are correlated with cell growth arrest or re- proteins are necessary for optimal DNA primase activ- duced growth rate,accumulation of dumbbell-shaped ity (FOIANIet al. 1989a).However, the inability to cells and inability to synthesize appropriate amounts purify each primase subunit in an isolated form in any of high molecular weight DNA molecules (FRANCES- eukaryotic system has until now limited the possibility CONI et al. 1991).

Genetics 133 183-1 91 (February, 1993) 184 M. P. Lo1nghese et al. TABLE 1 (Table 1). MATa pril-1, MATa pri2-1 and MATa pri2-2 meiotic segregants from the obtained diploid strains were S. cerevisiae strains then back-crossed to TS72, A16 and F402, respectively, to originate the corresponding prillpril or pri2/pri2 strains, Strain Genotype and to TD28 togive rise to PRIllpril or PRI2/pri2 strains. DGL2-I Ca MATa met4 lysl Rich medium YPD, synthetic medium SD and synthetic TD28" MATaura3-52 canlinol complete medium with amino acids and bases omitted as TS72a MATaura3-52 inolcanlpril-1 specified, were as described by SHERMAN,FINK and HICKS cs33a MATaura3-52 inolcanlpril-2 (1 986). L-Canavanine sulfate was added to synthetic com- A16a MATaura3-52 pri2-1canlinol plete medium lacking arginine at the final concentration of F402" MATaura3-52 canlinolpri2-2 30 pg/ml. Sporulation media and conditions were as de- TS 1-4b MATaura3-52 inol canl poll-I scribed by LUCCHINIet al. (1 978). DGL273-3Db MATaura3-52 lysl canl cdcl7-1 Fluctuation test: For the recombination assaywe used DGL277-5Bb MATaura3-52 lysl canlinol cdc 17-2 four pril-1, four pri2-1, three pri2-2, one pril-2 and four DGL278-9Ab MATaura3-52 lysl inolhpr3 PRIlPRI2 (wild-type) strains constructed as described 344-109DC MATaura3-52 trpl adel-I01 leu2- above, which were all Leu- His- Ura+ Trp+ andwere shown I1Z::URA3::leuZ-k his3- by Southern analysis to carry both the LEU2 and the HIS3 513::TRPI::his3-537 duplications as depicted in Figure 1A. Analysis of the CANl 344-115BC MATaura3-52 trpl leu2- to canl mutation rate was performed on three CANl pril- I I 2::URA3::leu2-k his5 l, one CANl pril-2, three CANl pri2-l, one CANl pri2-2, 513::TRPI::his3-537 one CANl poll-1, one CANl cdcl7-1, one CANl cdc17-2, one CANl hpr3 and 10 CANl PRIl PRI2 POL1 (wild-type) Described in FRANCESCONIet al. (199 1). strains. According to the procedure described by AGUILERA Described in LUCCHINIet al. (1990). and KLEIN (1988), with minor modifications, single cells Described in ACUILERAand KLEIN (1988). were allowed to form colonies (approximately 2-3 X 10' cells/colony) on YPD plates at 25" (wild-type,pril-1, pril-2 We have now further characterized these mutants and pri2-1 strains), 28' (wild-type and pri2-2 strains for the by studying their behavior with respect to other proc- recombination test) or 37" (wild-type and pri2-2 strains for essesinvolving DNA metabolism,including mitotic the mutation test). Growth temperatures were chosen as the recombination, spontaneous mutation frequency and ones providing the higher recombination or mutation rates meiosis, all of which might be affected by defects in in the mutants, based on preliminary tests.Five to eight independent colonies for each strain were resuspended in 1 the DNA replication machinery. Moreover, we report ml of water and plated on selective SD -His and SD -Leu a preliminarycharacterization of thepola-primase media (approximately 3 X lo5 cells/plate) for the recombi- complex in the different mutants that provides some nation test, on SD + canavanine (approximately 1 X IO' new insights about protein-protein interactionsamong cells/plate) for the mutation test, and on complete synthetic the pola-primasepolypeptides and the role of the medium (approximately 3 X 10' cells/plate) to determine the exact viable title of each cell suspension. Plates were primase subunits in supporting enzyme activity. incubated at 25" and colonies were counted after 5 days. Leu+ or His+ recombinant clones were then replicated on MATERIALS ANDMETHODS SD -Ura and SD -Trp, respectively, to distinguish between reciprocal recombination and gene conversion events. Re- Strains and media: The original pril and pri2 mutant combination and mutation rates (recombinantsor mutants/ strains TS72, CS33, A16 and F402 and their isogenic wild- cell/generation) were calculated for each strain according type strain TD28 (Table 1) were used for immunoprecipi- to LEAand COULSON(1948). No significant differences were tation and immunopurification of the pola-primase com- observed in either recombination or mutation rates among plex. Strains used to determine intrachromosomal recom- different wild-type strains, even if pregrown at different bination rate and CANl to canl mutation rate were con- temperatures, or among different strains carrying the same structed as follows. First, the isogenic mutant strains TS72, pril or pri2 conditional allele. CS33, A16 and F402 were crossed tostrain 344-1 15B Biochemical methods: Preparation of yeast crude ex- (Table l), togenerate diploid strain DML82, DML83, tracts, polyacrylamide gel electrophoresis in the presence of DML84, DML85, respectively. One pril-1, pril-2, pri2-1or sodium dodecyl sulfate (SDS-PAGE), Western blot analysis, pri2-2 meiotic segregant from each of these diploids was and immunochromatographic purification of the pola-pri- then crossed to strain 344-109D (Table 1) to obtain, respec- mase complex with the y48 monoclonal antibody (Mab) tively, the diploid strains DML150, DML148, DML96 and covalently coupled to Protein A-Sepharose 6B (Pharmacia) DMLlO2. Strains with the appropriate genotype were cho- were as previously described (PLEVANIet al. 1985). Immu- sen among the meiotic segregants from this second round noprecipitation of the pola-primase complex from wild-type of diploids to carry on the two assays described in the next and mutant cells was achieved by adsorption for 2 hr at 4" section. CANl poll-1 and CANl cdcl7-1 strains were ob- of 25 mg of protein in the corresponding extracts to 0.15 tained as meiotic segregants from crosses TS1-4 to 344- ml of a 1: 1 dilution of Mab y48-Protein A-Sepharose 6B in 115Band DGL273-3D to 344-115B, respectively, while breaking buffer (50 mM Tris-HCI at pH 8.0, 100 mM NaCI, CAN1 hpr3 and CANl cdcl7-2segregants were derived from 0.5 mM EDTA, 5 mM MgCln, 1% dimethyl sulfoxide, 1 mM crosses DGL278-9A to 344-109D and DGL277-5B to 344- phenylmethylsulfonyl fluoride, 1 pg/ml of pepstatin A, leu- 109D, respectively (Table 1). Strain TS1-4 is isogenic to peptin and chymostatin). After centrifugation for 2 min in TD28, while strains DGL273-3D, DGL278-9A and an Eppendorf centrifuge, the resin has been washed three DGL277-5B are strictly related to TD28 (LUCCHINIet al. times with 5 ml of breaking buffer and divided in several 1990). Diploid strains for sporulation analysis were con- aliquots, correspondingto 2 mgof protein in the initial structed by crossing first TS72, A16 and F402 to DGL2-IC extracts. Each aliquot was then resuspended in 0.080 ml of Yeast DNA Primase Mutants I85 DNA polymerase or DNA primase reaction mixtures and A enzymatic activities in the immunoprecipitates were meas- leu 24 URA3 Iou2.112 Chr 111 @RZ* tzBKx3 ured as previously described, with [3H]dATP (750 cpm/ tIxI pmol) as the labeled substrate (PLEVANIet al. 1985). The Chr V -%- immunological reagents used for Western blot analysis to hS3413 ARSI.lRP1 hsJ.537 detect pola were a mixture of anti-pola Mabs (LUCCHINIet Chr XV P al. Irn 1 1985). Affinity purified rabbit polyclonal antibodies Chr N m raised against trpE-PRI2 or trpE-PRI 1 fusion proteins, pro- duced in Escherichiacoli and containing, respectively, the B carboxyl-terminal 4 19 amino acids of p58 or the carboxyl- Rate (Xloq LW+ nw " terminal 307 amino acids of p48were used to detect p58or &rnples' nia+ LW* Ura+tUn- Trp*/Tw p48 on Western blots. These antibodies specifically recog- wild-lype 26 7[25"-28O] 5 12 4 13 11 nize either p58 or p48 on Western blots of both immuno- purified pola-primase complex and yeast crude extracts pn 1-1 22 [25O] 161 (~21) 445 (x36) 19 25 (SANTOCANALEet al. 1993; this study). pn 1-2 8 [25'] 181 (~24) 306 (x25) 1 5 35

pnbl 137 (~18) 73 (6)1 1 16 RESULTS 29 [25O] 1 1 16 pn2-2 20 [28O] 34 (x4 5) 25 (x2) Conditional priZ and pri2 mutations lead to en- hanced rates of intrachromosomal mitotic recombi- FIGURE1.-Experimental scheme to measure the effect of pril and pri2 mutations on intrachromosomal recombination. (A) The nationand mutation: An increase in therate of common basic genetic features of all the strains used for this study mitotic recombination and mutation has been ob- are shown (see text for details). (B) The rate of recombination at served as a consequence of ts mutations in a number LEU2 and HIS3 in wild-type, pril and pri2 strains is given, as well of DNA synthesis genes, including CDC9 (DNA ) as the ratio (Ura'/Ura- and Trp+/Trp-) between gene conversion and popout events at each locus. (GAME,JOHNSTON and VONBORSTEL 1979), CDC8 Total number of independent clones analyzed for each pril or (thymidylate ), POLlICDCl7 (DNA polymerase pri2 allele. Growth temperature is indicated within brackets. a),and POL3ICDC2 (DNA polymerase 6) (HARTWELL ' Each entry represents the average value of the rates obtained and SMITH1985; ACUILERAand KLEIN 1988). for each different strain carrying the same pril or pri2 alleles To determine whetherdefects in the DNA primase according to the fluctuation test of LEAand Cou~so~(1948). The subunits would also affect mitotic recombination, we standard error was always <20%. Numbers in parenthesis indicate increase over the wild-type rate of recombination. used a genetic system capable of measuring both gene conversion and reciprocal recombination events, pre- 10 tetrads from each of them were analyzed and we viously described by ACUILERAand KLEIN (1988). observed a 2:2 segregation of high frequency of Leu+ Briefly. the system is basedon haploid strains carrying and His+ papillae in all the tested tetrads. This hyper- heteroallelic duplications of the LEU2 gene on chro- recombination phenotype always cosegregated with 1110some Ill and of the HIS3 gene on chromosomeXV the pril or pri2 mutations, as exemplified in Figure 2 (Figure 1A). The URA3 marker is located between the for the pril-1 and pri2-1 alleles, and it was not ob- leu2-112 and theleu2-k alleles, while the TRPl marker served in tetrads fromPRIIIPRIl PRI2/PRI2 control separates the his3-513 and the his3-537 alleles, and strains. These data indicated that the observed hyper- the corresponding chromosomal loci carrymutant recombination phenotype was dueto the primase ura3 and trpl alleles, respectively. Recombinants are mutations under analysis. To quantify the effect of selected as Leu+ or His+ clones, which are then ana- the pril and pri2 alleles on recombination, we per- lyzed by replica plating to distinguish between differ- formed a fluctuation test as described in MATERIALS ent recombination events involving the repeated se- AND METHODS. As shownin Figure IB, allof the quences at each locus: (i) reciprocal recombination, mutations, todifferent extents, cause a significant that leads to loss of the URA3 or TRPl marker (Leu+ enhancement in recombination rates at both loci. Ura- and His+ Trp- clones) and (ii) gene conversion Quantitative differences among different mutantsare events (Leu+ Ura+and His+ Trp+ clones). The dupli- positively correlated with the effect of the mutations cations were introducedinto a pril or pri2 back- on growthrate at the temperatureused for therecom- ground by two rounds of crosses (see MATERIALS AND bination assay (FRANCESCONIet al. 199 1). Thus, the ts METHODS) involving the isogenic mutant strains and pril-1 strains and thecspril-2 strains show the highest two wild-type strains carrying the duplications, which recombination rate (20-30-fold increase over the are genetically related to each other and show nearly wild-type rate) and grow more slowly at 25" than the identical rates of mitotic intrachromosomal recombi- ts pri2-1 strains (6-18-fold increase). In addition, the nation and spontaneous CAN1 to can1 mutation growth rate at 28" is nearly wild-type in the ts pri2-2 (ACUILERAand KLEIN 1988; our unpublished obser- strains, which show only a modest 2-4-fold enhance- vation). The second round of crosses gave rise to ment in recombination rate. We observed an increase diploid strains DML150, DML148, DML96 and in both reciprocal recombination and geneconversion DML102, that were heterozygous for onepril orpri2 events inall the mutants (Figure 1B). Whereas the allele and homozygous for the duplications. At least ratio of the frequencies for the two kinds of recom- 186 M. P. Longhese et al.

FIGURE2.-The pril-I and pri2-l ts phenotype is associated with high frequency of recombination. Tetrads from the diploid strains DML126 PRIl/PRII PR12/PR12 (lanes 1 and 2). DML150 priI-I/PRII PR12/PR12 (lanes 3 and 4) and DML96 PRII/PRII pri2-1lPR12 (lanes 5 and 6), all carrying homozygous LEU2 and HIS3 duplications, were dissected after sporulation and analyzed by spotting about 1 Os cells for each segregant clone on plates containing the indicated media. Strain DMLl26 was obtained by crossing one PR11 meiotic segregant from DML82 to strain 344-109D. Pictures were taken after 36 hours of incubation at 25" or 37" for YPD plates and after three days of incubation at 25" for SD -Leu and SD -His plates. Growth on SD complete medium was comparable to growth on YPD at the same temperatures. Two tetrads are shown in each lane and letters A, B. C. D indicate four spores from the same tetrad. bination events are very similar in wild-type and pri2 TABLE 2 strains, both pril mutations seem to favour gene con- Mutator phenotype of DNA primase mutants version, since the difference compared to wild type was significant at P < 0.01 by x2 analysis. CAN1 to ran1 Temperature mutation rate Mutations affecting mitotic recombination often Strain ("C) Samplesa (X I0")b have been observed to cause a mutator phenotype W ild-type 25-37Wild-type 80 I .0 (HARTWELL andSMITH 1985; AGUILERAand KLEIN pri1-l 25 24 7.5 1988). For this reason, we analyzed the effect of pril pril-2 25 I6 10.0 and pri2 mutationsspontaneous on rate of mutation pri2- I 25 32 6.7 of the of wild-type CAN1 gene to can1 alleles that confer pri2-2 37 8 4.3 resistance to the arginine analoguecanavanine, and a Poll-1 25 8 7.7 hpr3 25 8 6.1 fluctuation test was performed as described in MATE- cdcl7-l 25 8 7.0 RIALS AND METHODS. As a control, four different ts C~CI 7-2 25 8 3.3 poll alleles (poll-l, hpr3, cdcl7-l and cdc17-2) were a Totalnumber of independent clonesanalyzed for each pril or assayed in parallel. As summarized in Table 2, all the pri2 allele. b Fach entry represents the average value of the rates obtained 111utants' but One' showed a 6-10-fold enhancement for each different strain carrying the same pril or pri2 allele, ofCAN1 tocan 1 mutation rate compared to wild type, measured by the fluctuation test of Lea and CouIson (see MATERIALS when colonies were grown at 25 " before plating on AND METHODS). Standard error was always <20%. selective medium. Only the weak ts pri2-2 strain had diploid strains (see MATERIALS AND METHODS), carry- to be grown at 37" to observe any increase in the ing each tS pril and pri2 recessive mutationin the n1utation rate- Because the CAN1 to canl mutation heterozygous or homozygous state. Cells were grown rates were identical in wild-type strains grown at 25" in YPD at 250 and then transferred to liquid sporu- or at37", the 4-fold increase found in the pri2-2 lation medium at 250 or 280 (LUCCHINIet al. 1978). strain can be attributed to the mutation in the PR12 samples for DAPI staining.. of the nuclei were taken gene. at different times and sporulation frequency and spore PriI and pri2 ts mutations interfere with meiosis survival were determined after three days. and spore survival: The essential role of polcvin As summarized in Table 3, sporulation is severely premeiotic DNA synthesis has been clearly demon- affected in pril-l and pri2-l homozygous strains even strated (BUDDet al. 1989). It was therefore of interest at 25",a temperature that either doesnot alter (pri2- to determine whether the associated DNA primase, I)or causes only a slight delay (pril-l)in the rates of essential for mitotic DNA synthesis, was also involved mitotic growth and DNA synthesis (FRANCESCONIet in the meiotic process. For this purpose we compared al. 1991). The pri2-2 homozygous strain also exhibits sporulation frequencies and spore survival in isogenic a less severe defect in sporulation, as expected from Yeast DNA Primlase Mutants I87 TABLE 3 As summarized in Table 4, with the only exception of Effect of priZ and pri2 mutations on sporulation and spore rare pril-2pri2-2 segregants, whichwere severely survival affected in growth rate at any temperature, all the

~ ~ ~~~~ other double pril pri2 combinations had a lethal ef- S rulation No. of tetrads fect. These data addfurther evidence for in vivo Kquincy "ith viable (%) spores' interaction between the two subunits. No. of dissected A parallel biochemical approach allowed us to ana- Strain 25" 28" tetrads' 4 3 2 I lyze the effect of these mutations on the stability of PRll/priI-l 35 30 8 7 1 the corresponding gene products and on their ability pril-l/pril-l 8 1.5 7 232 to interact with one another and with pola. Immu- PR121pri2-I 36 3 1 8 7 1 pri2-llpn2-1 5 0 9 2214 noblot analysis of mutant crude extracts (Figure 4) PR121pri2-2 28 29 8 71 shows that pril-l and pri2-l alleles encode very labile pri2-21pri2-2 20 14 8 332 polypeptides that are barely detectable using our an- 'Tetrads were dissected from the 25" sporulated cultures. tibodies, whereas the pril-2 mutation does not signif- b Spore viability was assayed at 25" on YPD. icantly affect the level of the corresponding polypep- tide and p58 level is significantly lower than in wild- type in pri2-2 cells. These differences in protein sta- bility correlate well with the effect of the mutations on growth rate and DNA synthesis. The level of the other wild-type primase subunit was substantially un- affected in all the mutant extracts. To analyze the effect of the mutations on the inter- actions anlong the pola-primase complexpolypep- tides, we applied an immunochromatographic proce- dure, basedon the useof the anti-pola Mab y48, which allowsthe purification of the pola-primase com- plex from wild-type cells (PLEVANIet al. 1985). With this procedure, we immunopurified pola and the as- sociated polypeptides from the above pril and pri2 FIGUREJ.-DAPI stained nuclei in sporulating wiltl-type and pri2-l diploid cells. S;umples for staining with 4',6-dia1nidino-2- mutant extracts. As shown in Figure 5, the amount of phenylindole (DAH: -rRUEHEART, BOEKEand FINK, 1987) were p48 associated with pola is severely reduced in pril-I collected 4X hours after cell transfer to sporulation medium (see preparations, in accord with the increased lability of text for details). the mutated polypeptide (Figure 4), while the ratio the lesser effect of this mutation on cell growth and between the amount of p58 and pola polypeptides is DNA synthesis. All the unsporulated cells in the similar to the wild-type preparation. In contrast, the homozygous mutants arrested with a single nucleus, amounts of both the mutated p58 and the wild-type as illustrated for the pri2-l/pri2-1 strain in Figure 3. p48 associated with pola in pri2-l are bothhighly Moreover, the appearance of asciwas delayed by more reduced, even though the level of p48 was normal in than 12 hr for the homozygous strains compared to the crude extract (Figure 4). Similarly, the amount of the corresponding heterozygous strains at 25" (data pola-associated wild-typep48 in pri2-2 preparations is not shown), and a significant number contained invi- reduced from the wild-type and seems to be deter- able spores (Table 3). The latter finding suggests that, mined by the amount of mutant p58 recovered. even when meiosis and sporulation take place, some To further analyze the role of the two subunits in meiotic products are genetically defective. enzyme function, we tried to correlate primase activity Mutations in the PRIl and PR12 genes differently with the variable levels of the primase polypeptides affect protein-proteininteractions: Since p48 and observed in wild-type, pril-1 and pri2-l complexes. p58 polypeptides interact with each other, it could be Towards this end, we used the anti-pola y48 antibod- expected that specific missense mutations in one of ies, which do not inhibit either DNA polymerase or the two genes would suppress certain missense muta- primase activity (PLEVANIet al. 1985), to immunopre- tions in the other gene. Alternatively, combinations cipitate pola from wild-type and mutant extracts. The of conditional mutations in both genes might resultin immunoprecipitates were analyzed directly for DNA a more dramatic defect than would the single muta- polymerase and primaseactivities and by Western tions. In order to address this question, we crossed blotting. the pril and pri2 conditional nlutants in all the possi- The polypeptide compositionof the immunoprecip- ble pairwise combinationsand analyzed the phenotype itates was similar to that observed after immuno- of double pril and pri2 segregants from these crosses. chromatography, although, under these conditions, 188 M. P. Longhese et al.

TABLE 4 Lethality of pril pri2 double mutants

No. of tetrads with viable sporesb No. of dissected Strain tetradsa Strain 4 3 2

3 (NPD) "PRllbri2-2 pril-lPR12 -PRll pri2-l 8 2 (PD) 4 (TT) 2 (NPD) pri 1-2 PR12 -PRI1 pr12-2 IO PD3 (1 + 2TT) 4 (3TT + INPD) 3 (NPD) pril-2 PRI2

Tetrads were defined as parental ditypes (PD), nonparental ditypes (NDP) or tetratypes (TT) based on segregation of the ts or cs phenotypes, that were shown to correlate with the presence of pril or/and pri2 mutations by a complementation test. From this analysis,all the expected pril pri2 double mutants were unviable, with the only exception of three pril-2 pri2-2 segregants, which gave rise to very small colonies. a Tetrads were dissected from the 25" sporulated cultures. Spore viability was assayed at 25" on YPD.

wr#-lvp. pfll-1 pfll-2 pri2-2 pfl2-1 abc.be abC abC *bC ptm -

.- 4 p180

-7-cp48

FIGURE4.-Western blot analysis on crude extract from wild- FIGURE5.-Western blot analysis of proteins purified from wild- type and pril or pri2 cells. Crude extracts were prepared from cell type, pril or pri2 cell extracts by anti-pola monoclonal antibodies. cultures grown in YPD at 25". Each lane represents 100 rg of the Immunochromatography with Maby48 was performedon the indicated crude extract, analyzed by SDS-PAGE, transferred to crude extractsdescribed in Figure 4. Polypeptides present in 0.0 15 nitrocellulose filters and probed, respectively, with a mixture of ml samples of each Mab eluate were separated by SDS-PAGE, anti-pola Mabs (top row), or with anti-p58 (middle row) or anti-p48 transferred to nitrocellulose filters and probed, respectively, with a (bottom row) polyclonal antibodies (see MATERIALS AND METHODS). mixture of anti-pola monoclonal antibodies (lanes a), or with anti- The identity of the bands in the crude extracts isassessed by p58 (lanes b) or anti-p48 (lanes c) polyclonalantibodies. comigration with the corresponding polypeptides in the immuno- purified pola-primase complex, that was run on the same gel. mutants when incubation was performed at the tem- the recovery of the p48 polypeptide from bothmutant perature permissive for the mutants (25"). E. coli DNA polymerase I works poorly at 25". When the extracts was somewhat higher relative to the level of same assay was performed at 37" to optimize elonga- 1'58 than that shown in Figure 5, but stillhighly tion of RNA primers by E. coli polymerase, primase reduced compared to wild-type levels. Assummarized activity in the mutants was found to be reduced to 6- in Table 5, the amount of DNA polymerase activity 11% of the wild-type level. To confirm that this 5- was comparable in wild-type and mutant immunopre- fold reduction of primase activity at nonpermissive cipitates, in agreement with the wild-type level ofpola temperature was due to a lower efficiency of primer polypeptide observed on Western blotsfor each prep- formation, a two-step reaction was carried out, where mition. Due to the very low amount of DNA primase primer formation was allowed to occur for 20 min. at in theseimmunoprecipitates, the efficiencyof the 25" or 37", before elongation by E. coli DNA polym- direct assay measuring the synthesisof labeled erase I couldtake place. Under theseconditions, oligo(rA) primers on a poly(dT) template (PLEVANIet primase activityin the mutant immunoprecipitates was al. 1985) was below the level of detection. Therefore reduced by preincubation at nonpermissiveversus we employed the more sensitive combined assay meas- permissive temperature, that we attribute to temper- uring E. coli DNA polymerase I activity as a function ature-sensitivity of the primase enzyme. The two-step of primer formation by yeast DNA primase. Under reaction comparison also indicatesthat primase activi- these assay conditions (Table 5), DNA primase activity ties measured in either mutant cannot be considered was reduced to 50-60% of the wild-type level in the as background levels. In fact, residual activity is de- Yeast DNA Primase Mutants 189

TABLE 5 DNA polymerase and DNA primase activity in immunoprecipitates

DNA primase activity (cpm)

DNAOne-step polymerase reactiona Two-step reactionb activity (cpm) at Immunoprecipitate 25" 25" 37" 25" "* 37" 37" + 37" Wild-type 37,118 173,990 2,203 84,499 179,950 pril-1 37,14620,160 1,157 8,107 3,440 30,276 1,335 10,035 10,123 3,170 10,123 10,035 pri2-1 1,335 30,276 . Numbers given in the table represent the average value of two independent assays on each immunoprecipitate. Standard error was always

peratureconditions which allow sporulation in the same amount of mutated or wild-type p48, respec- mutants, the resulting tetrads consistently containin- tively, but either a normal amount of wild-type p58 viablespores. Such spore inviability mightbe ex- (pril-1)or alnlost none of the mutated polypeptide plained by improperchromosome segregation, In (pri2-I).Therefore, the level of primase activity cor- fkt, a high frequency of chromosome loss has been relates with the amount of p48, independently on the observed in other DNA replication mutants, such as presenceof p58, suggesting that the small subunit cdc2 and cdcl7 (HARTWELLand SMITH1985), and has plays a major role in determining enzyme activity. In beenattributed to accumulationof DNA damage this case, the temperature-sensitivity observed in the resulting from imperfect DNA replication. Unfortu- pri2-I immunoprecipitate,that contains wild-type nately, our attempts to compare the DNA content of p48, might be ascribed to an increased thermolability our wild-type and mutant strains during sporulation of the p48 protein in theabsence of p58.These by flow cytometryor radioactivelabeling did not assumptions are supported by our recent demonstra- provide a clear pattern, due to the rather inefficient tion that isolated wild-type p48 is sufficient for RNA mid asynchronous sporulation that occursin our wild- primer synthesis in vitro, and it is more heat-sensitive type strains and to clumping and swelling of the mu- than when associated with p58 (SANTOCANALEet al. tant cells. Therefore, although our data strongly im- 1993). plicate DNA primase in premeiotic DNA synthesis, as Further biochemical characterization of the wild- shown for the associated pola (BUDDet al. 1989), the type and mutant primase subunits,well as as the search final demonstrationof this point must await more for unlinked genetic suppressors of the pril and pri2 detailed analyses. conditional mutations, arein progress to confirm the From our data thus far, mutations in either one of proposed different roles of thetwo subunits in enzyme the two primase genes lead to similar genetic defects, activity andto better understand their interactions and all pairwise combinations of the pril and pri2 within the polar-primase complex and with other com- conditional alleles under study result in a lethal phe- ponents of the DNA replication machinery. Ilotype. These results provide further evidence for a We thank H. L. KLEINfor providing yeast strains and M. MUZI direct interaction between thetwo subunits of primase FALCONI, A.PISERI and C. SANTOCANALE for preparing the anti- in vivo;however, they do notprovide any information bodies used in thisstudy andfor their help in the biochemical about thepossibility of different roles or unique inter- experiments. Thiswork has been partially supported by grants from actions of the two polypeptides with each other, with theTarget ProjectsBiotechnology and Bioinstrumentation and Genetic Engineering, Consiglio Nazionale delle Ricerche, Italy and pola or with other components of the DNA replica- by grant SC 1-0479-C(A) from the EuropeanEconomic Community. t ion machinery. On the other hand,it is significant to know moreabout the nature and severity of the LITERATURE CITED liochemical defect caused by a particular mutation, whileanalyzing its effects on various DNA transac- AGUILERA,A., and H. L. KLEIN, 1988 Genetic control of intra- tions in vivo. Our biochemical characterization of the chromosomal recombination in Saccharomyces cerevisiae. I. lao- lation and genetic characterizationof hyper-recombination mu- primase subunits has shown that the pril-1 and pri2- tations. Genetics 119: 779-790. 1 geneproducts are present at low levels in yeast BROOKS,M., and L. B. DUMAS,1989 DNA primase isolated from crudeextracts from the corresponding mutants, the yeast DNA primase-DNApolymerase complex. J. Biol. whereasthe same extracts contain normal level of Chem. 264: 3602-3610. BUDD,M. E., K. D. WITTRUP,J. E. BAILEYand J. L. CAMPBELL, wild-type p58or p48 subunit, respectively.Immu- 1989 DNA polymerase I is requiredfor premeiotic DNA nochromatographic purification of polcv from these replication and sporulation but not forX-ray repair in Succhu- extracts, under conditions allowing isolation of the romyces cerevisiae. Mol. Cell Biol. 9: 365-376. intact pola-primase complex from wild-type extracts, FOIANI,M., A. LINDER,G. R. HARTMANN,G. 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