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MOLECULAR AND CELLULAR BIOLOGY, Sept. 1994, p. 6039-6045

Vol. 14, No. 9

0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Mutation of the Gene Encoding Protein Kinase C 1 Stimulates Mitotic Recombination in Saccharomyces cerevisiae KIMBERLY N. HUANG AND LORRAINE S. SYMINGTON* Institute of Cancer Research and Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032 Received 28 April 1994/Returned for modification 17 May 1994/Accepted 8 June 1994

We have isolated a recessive allele of the yeast protein kinase C gene (PKCI) which promotes an elevated rate of mitotic recombination and confers a temperature-sensitive growth defect. The rate of recombination was increased between genes in direct repeat and at a series of heteroalleles and was dependent upon the RAD52 gene product. The mutant pkcl allele was sequenced and found to encode a single amino acid change within the catalytic domain. Osmotic stabilizing agents rescued the temperature-sensitive growth defect but not the hyperrecombination phenotype, indicating that the two traits are separable. This separability suggests that the PKCJ gene product (Pkclp) regulates DNA metabolism by an alternate pathway to that used in the regulation of cell lysis. The regulation of recombination is a previously unidentified role for Pkclp.

Protein kinase C (PKC) plays a critical role in the regulation of growth and proliferation in all eukaryotic cells (reviewed in reference 30). Two PKC homologs in the yeast Saccharomyces cerevisiae are encoded by the essential gene PKCJ (28) and the nonessential gene PKC2 (47). Deletion of PKCJ results in cell lysis (26, 27). Two temperature-sensitive (ts) alleles of PKCJ and one Ca2+-dependent allele were isolated by Levin and Bartlett-Heubusch (26), and additional alleles were found to be isogenic with clylS, a cell cycle mutant (34), and hypo2, a hypo-osmolarity-sensitive mutant (45). Several components of a Pkclp-regulated kinase cascade have been identified in yeast cells. These were isolated on the basis of their ability to suppress lysis in a strain deficient in either Pkclp or another component of the Pkclp-governed pathway (14, 23, 24, 25). Components of this cascade include members of the mitogenactivated protein kinase and mitogen-activated protein kinase kinase family, which have been implicated in PKC-mediated responses in mammalian cells (21, 27). Mutations that affect the rate of mitotic recombination have been isolated in numerous genetic screens. Members of the RAD52 epistasis group, which show decreased rates of recombination, were isolated by their increased sensitivity to X rays (8). Esposito et al. (6) used a haploid strain disomic for chromosome VII to isolate mutants with altered rates of interchromosomal recombination. Most of these mutants have not been well characterized HPR5. (SRS2), which encodes a DNA helicase (1, 38), and TOP3, a homolog of bacterial type I topoisomerases (52), were identified in screens for mutants with increased rates of recombination between repeated sequences. In addition, mutation of either TOPI or TOP2 confers a hyperrecombination phenotype specific for rDNA sequences (5). Most of the cdc mutants that exhibit S-phase arrest have also been shown to have hyperrecombination phenotypes (10, 11). To date, most hyperrecombination mutants define genes directly involved in DNA metabolism. It is likely that defects in proteins involved in DNA metabolism result in the accumulation of DNA lesions that are repaired by a recombinogenic mechanism.

Because the genetic screens used to isolate hyperrecombination yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with elevated rates of recombination. Here we describe a directrepeat recombination assay that uses the yeast ADE2 gene and the isolation of an allele of PKC1 that confers a hyperrecombination phenotype. To our knowledge, this is the first study to indicate that Pkclp regulates DNA metabolism.

MATERIALS AND METHODS Media and growth conditions. Media for yeast growth were prepared as described by Sherman et al. (44). YEPD medium (1% yeast extract, 2% Bacto Peptone, 2% dextrose, and 2% agar for plates) was used for nonselective growth. Osmotic stabilizing agents (1 M glucose, I M sorbitol, 0.5 M NaCl or 0.1 M CaCl2) were added to YEPD medium as indicated. Nutritional requirements were determined on synthetic medium lacking one amino acid or nucleic acid base (2% dextrose, 0.67% yeast nitrogen base, 2% agar). Selection for Ura- cells was on synthetic complete medium containing 5-fluoroorotic acid (5-FOA; 1 mg/ml) and uracil, (40 ,ug/ml). Yeast strains were grown at 24, 30, or 37°C as appropriate for each experiment. Escherichia coli strains were grown at 37°C in LB medium or LB supplemented with 100 ,ug of ampicillin per ml. Yeast transformation was performed by the lithium acetate method (15). Sporulation of diploids and dissection of tetrads were done as described by Sherman et al. (44). Construction of yeast strains. A complete list of the yeast strains used in this study is given in Table 1. Most of the strains were derived from W303-1A or W303-1B (50). An Ade+ derivative of W303-1A, YKH10, was made by one-step gene replacement (41) using a DNA fragment containing the ADE2 gene. The ade2-n::URA3::ade2-a construct was made as follows. The mutation in the 5' end of the ADE2 gene was introduced by digestion of a plasmid-borne ADE2 gene with the restriction enzyme AatII and treatment with T4 DNA polymerase to remove the 4-bp overhang. After ligation, the 3' mutation was created by digestion with NdeI and treatment with the Klenow fragment of DNA polymerase I to fill in a 2-bp overhang. After ligation and transformation, the resulting ade2 DNA was transferred to the yeast integration vector pRS306 (46) to

* Corresponding author. Mailing address: Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 West 168th St., Room 912, New York, NY 10032. Phone: (212) 305-4793. Fax: (212) 305-1741.

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HUANG AND SYMINGTON

MOL. CELL. BIOL. TABLE 1. Yeast strains used in this study

Strain

Genotype

W303-1A W303-1B YKH10 YKH12a YKH12ot YKH29a YKH29cx YKH74 DL454 YKH41 W838-24D W838-15B YKH22 YKH23 YKH60

MATa ade2-1 leu2-3,112 his3-]1,]5 canl-100 ura3-1 trpl-l MATot ade2-1 leu2-3,112 his3-1J,J5 can1-100 ura3-1 trpl-l AL4ATa leu2-3,112 his3-11,15 can 1-100 ura3-1 trpl-l MA Ta ade2-n::URA3::ade2-a leu2-3,112 his3- l,l5 canl-100 ura3-1 trpl-1 MATot ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 canl-100 ura3-1 trpl-l MATa pkcl-4 ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 cani-100 ura3-1 trpl-1 MATot pkc1-4 ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 canl -100 ura3-1 trpl]MATot pkcl-2::HIS3 ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 canl -100 ura3-1 trpl-] MATa leu2-3,112 trpl-1 ura3-52 his4 can] mpk]A::TRPJ MATaL ade2-n::URA3::ade2-a leu2-3,112 his3-1l,]5 can] ura3 trpl-1 mpklA::TRPI AATa ade2-1 leu2-3,112 his3-]],]5 canl-100 ura3-1 trpl-] radl::LEU2 rad52::TRPJ MAToa ade2-1 leu2-3,112 his3-11,15 can1-100 ura3-1 trpl-1 rad52::TRPJ MA Ta pkc1-4 rad52::TRPI ade2-n::URA3::ade2-a leu2-3,112 his3-]],15 can 1-100 ura3-1 trp]-] MATa rad52::TRPI ade2-n::URA3::ade2-a leu2-3,112 his3-11,]5 can]-100 ura3-1 trpl-] MATa radl::LEU2 rad52::TRPI ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 canl-100 ura3-1 trpl-l MATa pkc]-4 radl::LEU2 rad52::TRPI ade2-n::URA3::ade2-a leu2-3,112 his3-]l,l5 can]-100 ura3-1 trpl-] MATa radl::LEU2 ade2-n::URA3::ade2-a leu2-3,112 his3-ll,l5 can]-100 ura3-1 trpl-] MATa pkcl-4 radl::LEU2 ade2-n::URA3::ade2-a leu2-3,112 his3-11,15 canl-100 ura3-1 trpl-] MATot lys2 MATot pkc1-4 MA Ta pkcl-4 MAToh ade2 ade5 can] leul-12 trp5-d his7-2 tyrl-] Iys2-1 metl3-d ura3 MATa ade2 cyh2 leul-c trp5-c his7-1 tyrl-2 lys2-2 metl3-c ura3-1 MATa pkc1-4 ade2 cyh2 leul-c trpS-c his7-1 tyrl-2 Iys2-2 met]3-c ura3-1 MATot pkc1-4 ade2 ade5 can] leul-12 trp5-d his7-2 tyrl-] lys2-1 metl3-d ura3

YKH61 YKH62 YKH63 SJR13 YKH25 YKH27 REE209 REE218 YKH30 YKH31

create pKH9. This plasmid was linearized by digestion with BglII and used to transform the yeast strain YKH1O to uracil prototrophy. Transformants were analyzed by Southern analysis to ensure integration at the ADE2 locus (48). This strain was designated YKH12a. YKH12ot was made by backcrossing YKH12a to W303-1B and dissecting the resulting diploid to obtain a derivative with the opposite mating type. Mutagenesis of YKH12a was performed using a Stratalinker 2400 UV source (Stratagene) to irradiate cells to 20 to 80% survival. YKH29a was the result of mutagenesis of YKH12a. YKH29ot was derived by backcrossing YKH29a to YKH12a and sporulating the diploid to obtain a MTot segregant with the pkc]-4 allele. YKH74 was made by introducing a HIS3 marker downstream of the coding region of the pkc1-2 allele. The pkc]-2:: HIS3 construct was integrated at the PKC] locus of the yeast strain YKH12oc by one-step gene replacement (41). YKH41, containing a disruption of the MPK] gene, was made by crossing DL454 to YKH12ot and sporulation to recover Trp+ spores containing the ade2 direct repeat construct. The resulting haploids were backcrossed twice more to YKH12a or YKH12ot to produce YKH41. YKH22 and YKH23 were made by crossing YKH12a and YKH29a, respectively, to W838-15B and sporulation to create the haploid progeny with the genotypes indicated in Table 1. YKH60 and YKH62 were made by crossing YKH12cx to W838-24D and sporulation to create the haploid progeny with the genotype indicated. YKH61 and YKH63 were made by crossing YKH29oc to W838-24D and sporulation to create haploid progeny with the desired genotype. YKH30 and YKH31 were made as follows. YKH29a was crossed to SJR13, and the resulting diploid was sporulated. Spores prototrophic for LEU2, HIS3, LYS2, and TRPI were identified. These isolates, YKH25 and YKH27, were crossed to REE218 and REE209, respectively. Haploid progeny of these

Source or reference

50 50 This study This study This study This study This study This study 24 This study 29 29 This study This study This study

This study This study This study S. Jinks-Robertson This study This study R. E. Esposito R. E. Esposito This study This study

crosses which contained the auxotrophic markers of REE218 and REE209 were backcrossed to REE209 and REE218 three times to create YKH30 and YKH31. Cloning of the pkcl-4 allele. The pkc1-4 allele was transferred to a plasmid by gap repair (33). A plasmid containing a portion of the PKCJ gene on the vector YCp5O (39) was digested with restriction endonuclease BamHI to remove DNA encoding the catalytic domain and a segment of the 3' noncoding region. This fragment was used to transform a Uraderivative of YKH29a to uracil prototrophy. Plasmid DNA was recovered from the resulting transformants and the insert DNA was used to reconstruct an intact PKC] coding region. Although the resulting plasmid was capable of rescuing the temperature sensitivity of YKH29a, when a HIS3 marker was inserted downstream of the coding region and this DNA was used to replace the PKC] locus of YKH12a (41), the resulting strain was ts. Since introduction of HIS3 at the same position of the wild-type PKC] gene did not confer a ts phenotype, we reasoned that the gap repaired DNA contained the pkcl-4 allele. This was confirmed by sequence analysis. DNA sequence analysis. DNA sequencing was performed by the method of Sanger et al. (42), using the Sequenase kit (United States Biochemical). Determination of recombination rates. To determine the rate of adenine prototroph formation, cells were plated at the temperature indicated on either YEPD plates or YEPD supplemented with 100 mM CaCl2. Seven colonies were picked from each plate and dilutions plated on medium lacking adenine to determine the number of Ade+ cells and on YEPD to determine the total number of cells in the colony. The median Ade+ frequency was used to determine the rate of recombination per cell per generation (22). Each experiment was repeated three times, and the median rate for each strain is given in Tables 2 and 4. The rate of resistance to 5-FOA was determined in a similar manner, except with plating on syn-

PCK1 MUTATION STIMULATES RECOMBINATION

VOL. 14, 1994

A.

6041

B. Red, Ura+

pKH9

(Ndel)

AatI Bglll

NdeI

Popout

ADE2

(Aatll)

Gene Conversion

I

White, Ura+

* I

(Ndel)

(Aatll)

White, Ura

FIG. 1. (A) Construction of the recombination substrate. The mutations were introduced at the NdeI and AatII sites of the ADE2 gene. pKH9, containing the ade2 construct on the yeast integrating plasmid pRS306 (46), was digested with BglII and used to transform an Ade' derivative of the strain W303-1A (50) to uracil prototrophy. The resulting strain, YKH12a, contains two ade2 heteroalleles in direct repeat at the endogenous ADE2 locus. (B) Recombination pathways. Recombination between the two ade2 genes can produce a wild-type copy of ADE2. This can occur by a pop-out mechanism, resulting in loss of the intervening URA3 gene, or by gene conversion, in which the URA3 gene is retained. Either of these events can occur by an intrachromosomal mechanism (shown) or with the participation of a sister chromatid.

thetic medium containing 5-FOA instead of synthetic medium lacking adenine. For this experiment, the strains YKH12a and YKH29a were initially grown on YEPD medium at 30°C. To determine the percentage of Ura+ events, YKH12a and YKH29a were plated on YEPD medium at 30°C. Twenty-five colonies per strain were picked, and a dilution was plated onto medium lacking adenine. At least 18 Ade+ colonies were picked and tested for uracil prototrophy to determine a percentage of Ura+ events for each of the 25 isolates per strain. The median percentage of Ura+ events for each strain is given in Table 3. To determine the rate of heteroallelic recombination, the diploid strains REE209/REE218 (wild type) and YKH30/ YKH31 (pkcl-4/pkcl-4) were grown to mid-log phase in liquid YEPD supplemented with 100 mM CaCl2 at the temperatures indicated in Table 5. Seven cultures of each strain were analyzed at each temperature, and samples were plated to determine the frequency of prototrophy of the markers indicated. Again, median values were used to determine rates of recombination per cell per generation, and the experiments were repeated three times to generate the values shown. RESULTS Experimental system. To identify yeast recombination mutants, we developed a colony sectoring assay which uses two heteroalleles of the ade2 gene (49) in direct-repeat orientation at the ADE2 locus (Fig. 1A). One repeat contains a 2-bp insertion mutation at the 3' end of the gene (ade2-n), and the other contains a 4-bp deletion at the 5' end of the gene

(ade2-a). The repeats are separated by plasmid sequence and a copy of the URA3 gene. The mutations in ade2 cause the initial strain to form red colonies (36, 37). If recombination between the two alleles produces a wild-type ADE2 gene (Fig. 1B), a white sector will be formed within the red colony. Thus, the rate of sectoring indicates the rate of recombination within this strain, designated YKH12a (Fig. 2A). Identification of a hyperrecombination mutant. Strain YKH 12a was mutagenized by UV irradiation and screened for colonies with increased rates of sectoring. Of 20,000 colonies screened, 1 displayed a marked increase in the rate of sector formation (Fig. 2B). This hyperrecombination strain, designated YKH29a, also exhibited a ts growth defect. The ts and hyperrecombination phenotypes were recessive, segregated as single gene traits, and cosegregated in all tetrads analyzed, indicating that the two traits are likely due to a single mutation. To isolate the gene altered in YKH29a, a yeast genomic library (39) was introduced and transformants were selected at 37°C. Fourteen transformants were isolated and found to contain three different plasmids, all with overlapping DNA inserts. Subsequent subcloning and sequence analysis showed that the minimal complementing fragment contained the PKC1 gene. To confirm that the mutation in YKH29a was linked to PKC1, a HIS3 gene was inserted downstream of the PKCI coding region, and this construct was integrated at the endogenous PKC1 locus of YKH12a (41). When this strain was crossed to YKH29ot and 24 tetrads were dissected, the ts and hyperrecombination phenotypes segregated away from the His' marker in all tetrads analyzed, indicating tight linkage between the HIS3 gene and the mutation present in YKH29a.

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MOL. CELL. BIOL. TABLE 2. Rates of recombination at the ADE2 locus Strain

genotype Wild type

pkcl-4 pkcl-2 Wild type

F *

Yeast PKC1 Rat PKC1 (y) Rat PKC2 (p) Rat PKC8 Bovine PKCa Dros. PKC

885 414 401 411 398 400

...

F

F ...P F P F P F ... P F ...

...

...

LT L T L T L T L T LV

N L Q L Q L H L Q L

Y H H I H QL H

C S S T SC CT S C S C

F F F F F F

Q T E N R Q T P D R QT M D R QT K D H Q T V DR Q T M D[R

... ... ...

... ... ...

FIG. 3. Sequence alignment of PKC isozymes (20, 28, 32, 35, 40), with the altered amino acid in the pkcl-4 allele indicated. Dros., Drosophila.

rate

YEPD, 24-C YEPD, 24°C YEPD, 24°C

6.68 x 10-6 4.96 X 10-6

1 2.2 1.7

30°C 30°C 30°C 30°C

5.52 x 10-6 4.05 X 10-4 8.43 X 10-4 1.31 X 10-5

1 73 153 2.4

Wild type

Ca2+ 300C Ca2+ 300C

1.61 X 10-5 1.12 X 10-3

1 70

Ca2+ 370C Ca2+ 370C

2.89 X 10-5 5.28 X 10-3

1 182

Wild type

The pkcl allele from YKH29a was transferred to a yeast replicating plasmid by gap repair (33), and the DNA sequence was determined. A single nucleotide change, of serine 893 to a phenylalanine, was found (Fig. 3). This residue lies within subdomain IV of the family of serine-threonine protein kinases (9) and within the C3 domain of PKC isoforms (31). We have designated this allele pkcl-4. Rates of recombination were determined by measuring the frequency of adenine prototroph formation at different temperatures. The rate of recombination in the pkcc-4 strain was found to increase with elevated temperature (Table 2), consistent with impaired function at higher temperatures. It has been

Relative

YEPD, YEPD, YEPD, YEPD,

pkcl-4

FIG. 2. (A) Yeast strain YKH12a. The strain forms red colonies as result of the presence of the mutated ade2 gene. Recombination events which generate Ade+ cells are visualized as white sectors. (B) Strain YKH29a, containing the pkcl-4 allele in the YKH12a background. Cells were grown at 30°C on YEPD medium (44).

Rate/cell/ generation 3.00 X 10-6

pkcl-4 pkcl-2 mpkl pkcl-4

a

Conditions

reported that media containing osmotic stabilizing agents, including 100 mM CaCl2, 1 M glucose, 1 M sorbitol, or 0.5 M NaCl, are capable of suppressing the growth defects of other pkcl alleles and the lethality of a pkclI::LEU2 strain (26, 34, 45). When the pkcl-4 strain was plated on medium supplemented with any of these agents, the temperature sensitivity was suppressed but not the hyperrecombination phenotype (Table 2). This separability is likely to reflect the multiple functions for Pkclp in the cell. Pcklp is likely to govern both a lysis pathway which is rescued by osmotic support and a recombination pathway which is not. Further support for a dual-pathway model was found by measuring the rate of recombination in an mpklA::TRP1 strain. Mpklp is a downstream component of a Pkclp-regulated cascade modulating cell lysis. Deletion of MPK1 results in a temperature-dependent cell lysis defect similar to that observed in pkcl ts alleles (24, 26). The observation that an mpklA&::TRP1 strain displays wild-type levels of recombination (Table 2) supports the hypothesis that recombination is regulated by a separate mechanism to that used in cell wall metabolism. To determine if the increase in recombination was specific for the pkcl-4 allele, the ts pkcl-2 allele isolated by Levin and Bartlett-Heubusch (26) was introduced to the strain containing the ade2 direct repeat. The pkcl-2 allele is altered at residue 1023 of the catalytic domain. As shown in Table 2, this allele was also found to have an increased rate of recombination. The effect of these alleles on recombination is therefore likely to be due to a loss of function rather than an allele-specific gain of function. It is likely that the increase in the frequency of recombination events inpkcl strains is due to an increase in the frequency of DNA lesions rather than to a defect in the ability to repair these lesions. The pkcl-4 strain displays no increase in sensitivity to either UV or X irradiation, phenotypes which are commonly observed in DNA repair-deficient strains (data not

shown). Gene conversion and pop-out events are both increased in the pkcl-4 strain. Recombination between the ade2 repeats to generate a wild-type copy of the ADE2 gene can occur by two major mechanisms. Events in which the duplication remains intact are known as gene conversions. These can occur by an intrachromosomal interaction, by recombination between sister chromatids, or by a double-crossover event. Recombination events which result in the loss of one of the repeats and the URA3 marker are referred to as pop-outs. These can also occur

PCK1 MUTATION STIMULATES RECOMBINATION

VOL. 14, 1994

TABLE 4. The elevated recombination in pkcl-4 mutants is dependent upon RAD52'

TABLE 3. Gene conversion and pop-out events are both elevated in the pkcl-4 strain 5-FOAr Strain genotype

Wild type pkcl-4

% Ura+ of Ade+ events

50 45

6043

Strain genotype

Rate/cell/ generation

Relative

1.46 x 10-5 2.19 x 10-4

1 15

rate

by a variety of mechanisms, including intrachromosomal crossing over, unequal sister chromatid conversion, unequal sister chromatid exchange, single-strand annealing or replication mispairing. To determine if the increase in recombination in YKH29a occurs by a preferential increase in either gene conversion of pop-out events, the percentage of Ura+ events and the rate of resistance to 5-FOA were determined. If only gene conversion events are stimulated, the percentage of Ura+ events will increase significantly and the rate of 5-FOA resistance will remain similar to that of the wild-type strain. An increase in pop-out events is reflected in an increase in the rate of 5-FOA resistance. If pop-out events are uniquely increased, the percentage of Ura+ events will decrease sharply along with the increase in the rate of 5-FOA resistance. As shown in Table 3, both types of events are increased in the pkcl-4 background. There is a significant increase in the rate of 5-FOA resistance, yet the overall percentage of Ura+ events remains approximately the same as in the parental strain. The elevated rate of recombination in the pkcl-4 strain requires RAD52. Recombination at genes in direct repeat can proceed by two alternative pathways, one requiring the RAD52 gene product and the other requiring the RAD1 gene product (7, 19, 43, 51, 53). While Rad52p-mediated events proceed by a conservative pathway to produce gene conversion or crossover products, Radlp is involved in a nonconservative pathway specific for genes in direct repeat. To determine the mechanism of direct repeat recombination induced in the pkcl-4 strain, the rate of adenine prototroph formation was measured in radl, rad52, and radl rad52 backgrounds (Table 4). Recombination in the pkcl-4 strain was found to be dependent on the RADS2 gene product. The rate in a rad52 pkcl-4 double mutant was almost identical to that in a radS2 strain with a wild-type PKC1 gene. Deletion of RAD1 had little effect on the rate of recombination in the pkcl-4 background. Other members of the RAD52 epistasis group (8) which were tested for their effects on the rate of recombination in the pkcl-4 background include RAD54, RAD55, and RAD57. Mutations in these genes had no effect on the rate of recombination as judged by the sector frequency (not shown). This result was not unexpected as mutation in any of these three genes results in a hyperrecombination phenotype when assayed at genes in direct repeat (29). Heteroallelic recombination is elevated in the pkcl-4 mutant. To determine if the increase in recombination was specific either for genes in direct repeat or for events at the ADE2 locus, we used a second assay which measures mitotic interchromosomal recombination. Diploid strains containing heteroalleles of the leul, trp5, his7, tyrl, and lys2 genes at their endogenous chromosomal loci were constructed both with and without the pkcl-4 allele. The rates of prototroph formation at these loci were also found to be elevated in a temperaturedependent manner (Table 5). Therefore, mutation of PKC1 has a general effect on the rate of mitotic recombination.

Wild type pkcl-4 radl pkcl-4 radl rad52 pkcl-4 rad52 radl rad52 pkcl-4 radl rad52 a

Rate/cell/ generation

5.52 4.05 2.01 4.43 6.93 8.22 6.27 5.58

x 10-6 x 10-4 x 10-5 x 10-4 x 10-7

10-7

x X

10-8

x

10-8

Relative rate

1 73 3.6 80 0.13 0.15 0.01 0.01

All cells were cultured on YEDP plates at 30°C.

DISCUSSION We have developed an intrachromosomal recombination assay that uses ade2 heteroalleles in direct-repeat orientation at the ADE2 locus. The advantages of this assay are (i) recombination events are visualized by colony sectoring, (ii) recombination rates can be quantitated by the formation of Ade+ prototrophs, and (iii) both gene conversion and pop-out events can be monitored. Using this assay, we isolated an allele of PKC1 which promotes an increased rate of recombination and confers a ts growth defect. Because the addition of osmotic stabilizing agents to the medium suppresses the growth defect but not the hyperrecombination phenotype of a pkcl-4 strain, we suggest that the two phenotypes are controlled by different PKC1regulated pathways. This model is supported by our observation that an mpkl strain, which contains a mutation in a downstream component of the PKC1-regulated lysis pathway, does not display a significantly increased rate of recombina-

tion. There are at least three possible explanations for the increased rate of recombination in PKC1-deficient strains. First, because transcriptionally active sequences recombine at a higher rate than those that are inactive (50), and PKC in mammalian cells is known to stimulate production or activation of a number of transcription factors (3, 4, 13, 17, 30), it is possible that ADE2 expression is increased in pkcl mutants, promoting recombination. However, we have observed no difference in ADE2 transcript levels in wild-type and pkcl-4 strains at any temperature, indicating that increased transcription of target sequences is not responsible for the observed stimulation of recombination. A second possibility is that a prolonged S phase results in increased recombination rates. Cells containing the pkcl-4 allele accumulate at 37°C with small buds, indicating arrest in early S phase. However, the doubling rate of the pkcl-4 strain on plates containing osmotic stabilizing agents is nearly identical to that of the parental strain. Because osmotic stabilizing medium suppresses the growth defect without changing the relative rate of recombination, it is unlikely that the elevated recombination rates are due to a prolonged S phase. Third, it is possible that Pkclp regulates genes or proteins involved in DNA metabolism. Many genes involved in DNA metabolism display hyperrecombination phenotypes when mutated. These include the cell cycle genes CDC2, -6, -8, -9, -17, -45, -46, and -54 (10, 11), the genes HPR1, -4, and -5 (1), and the topoisomerase genes TOP1, -2, and -3 (5, 52). If one or more of these gene products requires functional Pkclp for optimal activity, reduced activity could result in the formation of recombinogenic lesions and lead to increased rates of recombination. Of these gene products, some are more likely

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HUANG AND SYMINGTON TABLE 5. Rates of mitotic heteroallelic recombination

Strain genotype

Wild type

pkcl-41pkcl-4

Temp (OC)

Trp+

Tyr+

Lys+

His+

24 24

2.15 X 10-6 6.70 x 10-6 3.12

1.87 x 10-6 7.55 X 10-6 4.04

1.57 X 10-7 9.60 X 10-8 0.61

2.00 x 10-7 5.26 X 10-7 2.63

1.51 x 10-7 3.72 X 10-7 2.46

30 30

2.79 x 10-6 2.14 x 10-5 8.93

2.43 x 10-6 2.23 x 10-5 9.16

1.63 x 10-7 2.76 x 10-7 1.69

1.04 x 10-7 7.57 x 10-7 7.25

1.87 x 10-7 9.89 x 10-7 5.30

37 37

3.28 x 10-6 1.28 x 10-4 39.1

2.60 x 10-6 9.18 x 10-6 3.53

2.12 x 10-7 7.30 x 10-6 34.4

7.13 x 10-8 4.07 x 10-6 57.1

1.37 x 10-7 3.57 x 10-6 26.1

Relative rate Wild type

pkcl-41pkcl-4 Relative rate Wild type

pkcl-4/pkcl-4 Relative rate

Rate/cell/generation

Leu+

candidates for regulation by Pkclp. For example, it is unlikely that the TOP] and TOP2 gene products are exclusive targets of Pkclp, as mutation in either of these genes results in elevated recombination only at the rDNA locus. Similarly, hprl mutants display elevated rates of pop-out recombination only, and this increase is partially dependent on RAD1 (2). The HPR4 and HPR5 gene products are also unlikely to be the exclusive targets of Pkclp, as hpr4 and hpr5 mutants display lower rates of recombination than pkcl strains. Because many of the cdc mutants show increases in recombination similar to that observed in the pkcl mutants, these gene products are the most likely candidates for regulation by Pkclp. A paradox arises as to why osmotic agents resolve an S-phase arrest, yet the strain displays a defect in DNA metabolism in the presence of these agents, suggesting an S-phase defect. It has been shown that the apparent S-phase arrest in pkcl-depleted cells is due to cellular lysis which occurs at the growing bud tip (27). Hence, osmotic agents prevent lysis in a manner independent of Pkclp kinase activity. Because pkcl strains are defective in aspects of DNA metabolism necessary for S-phase progression, it is likely that they do have S-phase defects, albeit without significant effect on the growth rate. Protein phosphorylation plays an important role in the regulation of a variety of cellular processes, including signal transduction, cell cycle progression, and transcription. Although the role of protein phosphorylation in DNA metabolism has been less well characterized, there are a number of protein kinases with potential roles in this process. Two protein kinases which regulate DNA repair in yeast are encoded by the HRR25 and DUNI genes. Inactivation of the Hrr25 kinase results in defective double-strand break repair, although spontaneous rates of mitotic recombination are not altered (12). The Dunl kinase is required for induction of ribonucleotide reductase in response to DNA damage (54). A third kinase which has a potential role in DNA metabolism is encoded by CDC5. Although cdc5 mutants arrest in M phase at the nonpermissive temperature, they display a hyperrecombination phenotype, indicating a potential role for Cdc5 in S phase as well (1, 10). In support of this observation, the CDC5 gene was isolated as a dosage-dependent suppressor of a dbf4 mutation (18). DBF4 is required for the initiation of chromosomal DNA replication. Last, mutation in the protein kinase gene DBF2 results in delayed DNA synthesis (16). ACKNOWLEDGMENTS We thank M. Rose for the library, D. Gottschling for the ADE2 plasmid, and D. Levin, R. E. Esposito, R. Rothstein, and H. Klein for strains. We also thank H. Klein, A. Mitchell, R. Rothstein, and I. B. Weinstein for critical reading of the manuscript.

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