ABSTRACT
Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation. In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13. AlthoughCDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells. Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level. Thus, incdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres. Furthermore, chromosome end structure remains protected in cdc13-4mutants. Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Δ double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y′ elements after 225 generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance. Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres. Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity.
The ends of linear eukaryotic chromosomes form a special structure, the telomere. The telomeric DNA-protein complexes are essential for chromosome stability (49). They protect chromosomes from degradation and end-to-end fusion (54) and ensure their complete replication (41). In most eukaryotes, telomeric DNA contains a simple, repetitive sequence with the strand running toward the end of the chromosome being rich in G residues. For some organisms the configuration of the chromosome ends has been defined exactly. In hypotrichous ciliates the double-stranded region is followed by a 12- to 16-nucleotide-long single-stranded (ss) 3′ overhang (22, 24), whereas mouse and human chromosomes contain ss termini of 45 to 200 nucleotides (36, 39, 60). In the yeastSaccharomyces cerevisiae the telomere repeats consist of 300 ± 75 bp of C1–3A/TG1–3 DNA. Detectable ss extensions of the G-rich strand are generated at telomeres specifically during S phase in a telomerase-independent process (11, 57, 58). A specialized enzyme, telomerase, performs synthesis of telomeric DNA by extending the 3′ end of the G-rich strand of the telomere. Telomerase activity in S. cerevisiae depends on at least four protein subunits (encoded byEST1, EST2, EST3, and CDC13/EST4) (28, 34) and the RNA component (encoded by TLC1) (52). All subunits are essential for telomerase function in vivo, although only the catalytic subunit EST2 and the RNA template TLC1 are necessary for in vitro activity (6, 8, 30). Deletion of most individual components of the telomerase complex leads to inactivation of telomerase and thereby to a decrease in telomere length and to replicative senescence (28, 34).
However, deletion of CDC13/EST4 leads to immediate cell cycle arrest and cell death (56). This phenotype is triggered by the accumulation of telomeric single-stranded DNA (ssDNA) that activates an RAD9-dependent G2 arrest (16). Therefore Cdc13p was proposed to provide protection of the telomere from nucleolytic degradation by DNA end binding. This role is consistent with the finding that Cdc13p binds ss telomeric DNA in vitro (29, 40) and binds exclusively to telomeric, but not to internal, C1–3A/TG1–3 repeat sequences (5). Very recently the DNA binding domain of Cdc13p has been mapped to amino acids 557 to 694. Heterologous expression inEscherichia coli of a small, CDC13-derived polypeptide containing this region results in a protein that binds, like the full-length Cdc13p, with high affinity to ss telomeric DNA (23). A single amino acid missense mutation within this region of Cdc13p causes thermolabile DNA binding, and consistent with the presumption that Cdc13p DNA binding is essential to protect chromosome ends, this mutant is temperature sensitive for growth (23).
Besides its role in chromosome end protection, Cdc13p is involved in recruiting telomerase to telomeric DNA.cdc13-2est mutant cells exhibit a senescence phenotype but can be rescued by expression of a Cdc13-2est–Est1 fusion protein (12). These data suggest that Cdc13p is essential for loading telomerase to the telomere and that this process is mediated via interaction with Est1p. Interaction of Cdc13p and Est1p has been shown by two-hybrid criteria. Additionally, hemagglutinin (HA)-tagged Cdc13p can be copurified with a glutathione S-transferase (GST)–Est1 fusion protein from yeast extracts if both proteins were overexpressed (45). Furthermore, Cdc13p seems to be involved in the accurate regulation of telomerase recruitment, as severalCDC13 mutations, not yet mapped at the genomic level, confer either elongated telomeres (41, 18) or shortened telomeres (18).
In S. cerevisiae the steady-state level of telomeric GT repeat tract length seems to result from a balance between telomere elongation and telomere shortening (37). Many proteins involved in telomere length maintenance have been identified already. A major factor involved in negative telomere length regulation is the Rap1 protein, which binds with high affinity to specific sequences within the telomeric GT repeat tracts (7). Unregulated telomere elongation is prevented mainly by Rap1p and its interacting partners Rif1p and Rif2p (21, 31, 59). It has been proposed that a negative feedback mechanism determines the exact number of Rap1p molecules bound to telomeric DNA and regulates telomerase activity (37, 38). Recently, a model has been suggested in which a special folded structure prevents telomere elongation (46). In this model, the formation of the folded structure of the chromosome end depends on the length of the GT repeat tract and on the number of bound Rap1p.
At least two pathways are involved in positive telomere length control in S. cerevisiae. One pathway involves Tel1p and the Mre11-Rad50-Xrs2 complex, and disruption of any of these genes results in stable shortened telomeres (47). A second pathway affecting positive telomere length regulation involves the Yku heterodimer, which is also an essential component of the nonhomologous end-joining pathway (2-4, 44). As shown by in vivo cross-linking experiments, Ykup binds directly to telomeric DNA (19). Yku mutant strains display short but stable telomeres, and the ss telomeric overhang of the G-rich strand, usually restricted to S phase in wild-type cells, is present in Yku− cells throughout the entire cell cycle (19).
Using a genetic approach we identified a new mutation inCDC13, designated cdc13-4, that is lethal in combination with a deletion of either subunit of the Yku heterodimer. The telomeres of cdc13-4 mutants shorten continuously for about 150 generations before eventually reaching a stable level comparable to the telomere length seen in Yku− mutants.cdc13-4 causes no senescence phenotype, and acdc13-4/rad52 double mutant is viable for at least several hundred generations. A cdc13-4/tel1Δ double mutant displays enhanced telomere shortening compared to either single mutant and Y′ element amplification after 225 generations of growth. Coimmunoprecipitations reveal that HA3-Cdc13-4p still associates with GST-Est1p when both proteins are overexpressed. In addition, in a cdc13Δ strain a Cdc13-4–Est1 fusion protein does not induce telomere elongation to the same extent as a wild-type Cdc13-Est1 fusion. The terminal chromosome configuration of cdc13-4 mutants seems, besides the telomere shortening, unchanged, since no ss G-rich overhang can be detected by native in-gel hybridization. Our data indicate that Cdc13p functions in telomere length regulation independent of its roles in chromosome end protection and telomerase recruitment.
MATERIALS AND METHODS
S. cerevisiae strains, media, growth conditions, and transformation.The strains used in this study are listed in Table1. Cells were grown at 30°C using yeast extract-peptone-dextrose (YPD), yeast extract-peptone-galactose, or selective media as described elsewhere (14). Screening for synthetic lethal mutations was performed on YPD plates (9). For counterselection plates, 5-fluoroorotic acid (5-FOA) (bts) was added to selective media at a concentration of 1 mg/ml as described previously (9). To examine telomere length and the senescence phenotypes of strains over many generations, colonies derived from freshly germinated spores were streaked on YPD plates. After 48 h of incubation at 30°C, single colonies were restreaked on fresh YPD plates. This procedure was repeated up to nine times. Single colonies from different generations were then used for overnight inoculation and treated for DNA preparation. Yeast transformation was performed by the lithium acetate method (50).
Yeast strains used in this studya
Plasmids.The plasmid pCH-YKU70 used for the synthetic lethality screen was constructed as follows: aXhoI/EcoRI fragment containing a functionalYKU70 gene was isolated from the plasmid pRS316-YKU70 (15) and blunted with Klenow enzyme. This fragment was then cloned into pCH1122 (26) linearized withSmaI. Expression of Yku70p from pCH-YKU70 was verified by complementation of the temperature sensitive phenotype of ayku70-deficient strain and by reconstitution of Yku heterodimer DNA binding activity in a gel retardation assay (15). The CDC13 expression plasmid pRS314-CDC13 was generated as follows: a 4.7-kb ApaI fragment containing 712 bp 5′ of the start codon, the entire open reading frame (ORF) ofCDC13, and 1,200 bp 3′ of the stop codon was isolated from the library plasmid GP2a. This fragment was ligated to pRS314 (51), linearized with KpnI/SacI, and blunted with Klenow enzyme. To generate the plasmid pRS314-cdc13-4 expressing the mutated CDC13 allele, a 900-bp DNA fragment was amplified by PCR from genomic DNA of mutant LDM29 by using the primers CDC13-ATG (5′-ACG TGT CGA CCC GGG ATG GAT ACC TAG AAG AGC CTG AG-3′) and CDC13-900 (5′-GAA ATA TTT CCC GGT AGA GGA GG-3′). The PCR product was subcloned into pZErO-2 (Invitrogen) and sequenced. A XhoI/NsiI fragment carrying thecdc13-4 point mutation was then excised from pZ-cdc13-4 and ligated to the vector pRS314-CDC13 digested withXhoI/NsiI. To generate several CDC13disruption constructs, pRS314-CDC13 was digested withXhoI/AatII, thereby deleting the entire ORF ofCDC13 except 57 bp at the 5′ end. This fragment was replaced by a marker cassette of either KanMX4 or URA3, resulting in plasmid p-cdc13Δ::KanMX4 or p-cdc13Δ::URA3, respectively. The plasmid pRS-cdc13-4-KanMX4 was generated for genomic integration of the cdc13-4 allele by linearizing pRS314-cdc13-4 with AatII, blunting it with Klenow enzyme, and inserting the KanMX4 marker cassette.
To generate CDC13-EST1 and cdc13-4–EST1 fusion constructs, the EST1 gene was amplified from genomic DNA from strain W303a using primers Est1SacI (forward: 5′-GAG CTC ATG GAT AAT GAA GAA GTT AAC G-3′) and Est1SalISmaI (reverse: 5′-GTC GAC CCC GGG TCA AGT AGG AGT ATC TGG CAC-3′). A C-terminal fragment ofCDC13 was amplified using primers Cdc13-P3 (5′-CTG GTG CCA GGC GTC AAT TGC-3′) and Cdc13-P4Sma (5′-ATC CCG GGC GAG GTG GGA ACG GCT CCG-3′) and cloned into plasmid pZErO-2. TheEST1 fragment was digested usingSmaI/HpaI and ligated into pZ-CDC13-P3P4Sma linearized with SmaI. The correct orientation of the construct was verified by restriction analysis. The plasmid was then cut with SacII/PstI, and the DNA fragment containing C-terminal CDC13-EST1 was isolated. pRS314-CDC13 was digested with SacII/NotI, and a 3.1-kb fragment containing the N-terminal part of CDC13 and theCDC13 promoter was isolated. Both fragments were then ligated to pRS314 NotI/PstI, resulting in pRS314-CDC13-EST1. To delete the HindIII vector site, pRS314-CDC13-EST1 was cut with PstI/ApaI, treated with T4 polymerase, and religated. The religated vector was cut withNotI/KpnI, and the CDC13-EST1 fragment was isolated and ligated to pRS316 NotI/KpnI to obtain pRS316-CDC13-EST1. The plasmid pRS316-cdc13-4-EST1 was generated by restriction of pRS316-CDC13-EST1 withHindIII and replacing the resulting internalCDC13 HindIII fragment by the corresponding cdc13-4 HindIII fragment. The correct orientation of the cdc13-4 HindIII fragment was checked by restriction analysis, and sequencing confirmed the single base pair exchange in pRS316-cdc13-4-EST1.
Gene disruption.The yku70-deficient strain KαL7 was generated by disruption of the YKU70 gene in K2348α as described previously (15). Gene disruption was verified by Southern blot analysis. To disrupt the CDC13gene, plasmids pRS314-cdc13Δ::URA3 and pRS-cdc13Δ::KanMX4 were digested with ApaLI andKpnI, and the resulting linear disruption construct was used to transform several diploid strains to Ura+ or G418 resistance (Table 1). Disruption of the CDC13 gene was verified by Southern blot analysis. The yeast strain BMY13 carrying a genomic integrated cdc13-4 allele was generated by transforming LDY50 using the ApaI/KpnI fragment excised from pRS-cdc13-4-KanMX4. The transformed cells were plated on synthetic-dextrose minimal plates lacking uracil and containing 200 mg of G418/liter. Colonies arising from these plates were screened by PCR for correct integration of the marker gene. To verify the integration of the cdc13-4 point mutation, a PCR fragment spanning the corresponding part of the CDC13 gene was amplified and sequenced. BMY14 (W303aαcdc13::URA3/cdc13-4::kanMX4 rad52Δ::His3MX6/RAD52) was generated by replacement of the RAD52 ORF in BMY13 by PCR-based gene disruption (55). Sporulation of BMY13 and BMY14 resulted in haploid spores carrying the cdc13-4 point mutation (BMY17) and double mutant cdc13-4/rad52Δ (BMY18), respectively. Strain BMY56 was generated by crossing BMY17 with W303a. This strain was propagated for several generations and then used to introduce either a tel1 or est2 deletion.TEL1 was deleted by PCR-based replacement of the entire ORF with a His3MX marker (BMY57), and the EST2 gene was replaced by the TRP1 selection marker (BMY58). Transformants arising after incubation on selective media were screened by PCR for integration of the disruption constructs. Both heterozygous strains were then sporulated, and tetratype tetrads BMY59 and BMY60 were used for growth studies and analysis of telomere length phenotypes. To analyze expression of Cdc13-Est1 fusion proteins in acdc13Δ strain, BMY62 was transformed with pRS-CDC13-EST1 or pRS316-cdc13-4-EST1 and sporulated on plates lacking uracil. BMY64 and BMY65 were isolated after tetrad dissection of BMY62+pRS-CDC13-EST1 and BMY62+pRS-cdc13-4-EST1, respectively.
Induced expression of HA3-CDC13, HA3-cdc13-4, and GST-EST1.For induced overexpression of HA3-tagged CDC13 andcdc13-4, the GAL1 promoter together with the HA3 tag was introduced in front of the genomic copy ofCDC13 in W303aα or cdc13-4 in BMY56. Integration of GAL1-HA3 was performed by PCR-based methods as described previously (32) using the HIS3MX6 marker for selection. Correct integration of the HIS3MX-GAL1-HA3 construct in the resulting strains HFY80 (HA3-CDC13) and HFY84 (HA3-cdc13-4) was verified by analytic PCR and sequencing of the PCR product. Expression of HA3-Cdc13p and HA3-Cde13-4p was analyzed by Western blotting using monoclonal anti-HA antibody 9F10 (Roche). The same PCR-based strategy was used to generate strains expressing GST::Est1 fusion protein under control of the GAL1 promoter. TheTRP1-GAL1-GST construct was introduced in W303aα, HFY80, and HFY84 resulting in the strains HFY81 (GST::EST1/EST1), HFY82 (HA3-CDC13/CDC13GST::EST1/EST1) and HFY86 (HA3-cdc13-4/CDC13GST::EST1/EST1). Correct integration was verified by analytic PCR and sequencing of the PCR product. Expression of GST::Est1p was analyzed by Western blotting using monoclonal anti-GST antibody (Sigma). Strains were grown on yeast extract-peptone media containing 2% galactose for induced expression of HA3-CDC13, HA3-cdc13-4, and GST::EST1. Strains HFY81, HFY82, and HFY86 were sporulated to generate haploid strains expressing the tagged Cdc13 and/or Est1 proteins. Tetrad analysis was performed on yeast extract-peptone plates containing galactose to allow expression of HA3-Cdc13p, HA3-Cdc13-4, and GST::Est1p. Spores expressing the tagged proteins were identified by marker analysis, and the resulting strains HFY81-8A (GST::EST1), HFY82-6B (HA3-CDC13), HFY82-4C (HA3-CDC13 GST::EST1), HFY86-3A (HA3-cdc13-4GST::EST1), and HYF86-9D (HA3-cdc13-4 GST::EST1) were verified by Western blotting.
Immunoprecipitation.Coimmunoprecipitation experiments to analyze the interaction of GST-Est1p-HA3-Cdc13p and GST-Est1p-HA3-Cdc13-4p were performed using strains HFY82-4C, HFY86-3A, HFY86-9D, and, as controls, HFY81-8A and HFY82-6B. Crude extracts were prepared as follows: yeast strains were grown overnight in YPGal, diluted to an optical density at 600 nm (OD600) of 0.2, and grown to an OD600 of 0.8 to 1.2 in yeast extract-peptone-galactose. Cells were lysed in 20 mM Tris (pH 8.0)–200 mM NaCl–1 mM EDTA–1 mM dithiothreitol–0.01% NP-40–10% glycerol with one protease inhibitor cocktail tablet per 5 ml (complete, Mini, EDTA-free; Roche) in a bead beater. After centrifugation the soluble protein fraction was diluted 1:1 with lysis buffer containing 1% NP-40 and 0.2% Triton X-100. Crude extract (1,000 μg) was incubated with monoclonal anti-GST antibody, clone GST-2 (Sigma), for 1 h at 4°C, and then G-Sepharose (Pharmacia) was added. After incubation for 1 h at 4°C G-Sepharose beads were collected by centrifugation and washed twice with lysis buffer containing 0.5% NP-40–0.1% Triton X-100, twice with lysis buffer containing 1% NP-40–0.1% Triton X-100, and twice with lysis buffer containing 450 mM NaCl. The beads were treated with 1,000 U of DNase I/ml in lysis buffer containing 1 mM MgCl2 and then washed twice with lysis buffer containing 450 mM NaCl and 350 mM potassium acetate. After 15 μl of Laemmli buffer was added, beads were heated 3 min at 95°C and the supernatant was loaded onto an 8% sodium dodecyl sulfate gel. Proteins were visualized by enhanced chemiluminescence Western blotting using anti-HA antibody 9F10 (Roche) and anti-GST antibody clone GST-2 (Sigma).
Synthetic lethality screen.Stationary phase cells of KαL7 carrying the plasmid pCH-YKU70 were mutagenized with 3% ethyl methanesulfonate (EMS) for 90 min resulting in 15.6% survival. After EMS treatment, cells were plated on YPD plates containing 4% glucose to facilitate development of the red pigment. Uniformly red colonies were colony purified three times. Those which remained stably red under nonselective conditions were tested for sensitivity to 5-FOA. To test whether 5-FOA-sensitive cells were dependent on YKU70expression rather than other components of the plasmid pCH-YKU70, the mutants were transformed with a second plasmid, pRS314-YKU70, expressing Yku70p and containing TRP1 for selection. As a control, mutants were transformed with pRS314. Mutants carrying pRS314-YKU70 or pRS314 were retested for their ability to form red-white sectors and their growth on 5-FOA. Out of 20,520 mutagenized cells, five mutants were clearly dependent on YKU70expression. These mutants were stably red on YPD and sensitive to 5-FOA if transformed with the pRS314 vector control, but they displayed red-white sectoring colonies and growth on 5-FOA after transformation with pRS314-YKU70.
Complementation of YKU70 dependence.The mutant LDM29 was transformed using a single-copy genomic yeast library (ATCC 77164) and plated on Trp− media. Out of 12,500 primary transformants, 15 plasmids were isolated leading to red-white sectoring colonies even after retransformation. In addition, these 15 plasmids enabled LDM29 cells to grow on 5-FOA-containing media, indicating that those cells were independent of YKU70 expression. Restriction analysis revealed the isolation of three different genomic fragments capable of complementing the dependence on YKU70. To identify the isolated fragments, the 5′ and 3′ ends of the fragments were sequenced using vector-specific primers.
Identification of the cdc13-4 mutation.The genomic mutation in LDM29 was mapped by gap repair (42). Plasmid pRS314-CDC13 was digested using different combinations of restriction enzymes. The resulting linear plasmids were transformed into LDM29. Generation of a functional CDC13 gene by gap repair results in cells independent of YKU70 expression, therefore displaying a red-white sectoring phenotype. Only cells transformed with pRS314-CDC13 with a XhoI/NsiI fragment spanning bp +57 to +830 of the CDC13 coding sequence deleted did not display red-white sectoring colonies and were sensitive to 5-FOA, indicating that a plasmid carrying the mutated allele of CDC13 was generated. To identify the mutation, a fragment corresponding to the mutated region in CDC13 was amplified by PCR from genomic DNA of LDM29 and was sequenced.
Yeast DNA extraction and analysis of telomeric DNA.Genomic DNA was isolated from 5- to 7-ml overnight cultures using the nucleon MiY DNA extraction kit (Amersham Life Science). For analysis of telomere length, genomic DNA was digested overnight usingXhoI and separated on a 1% agarose gel in 1× Tris-acetate-EDTA buffer. DNA was transferred to nylon membranes (HybondN+) by vacuum blotting using 0.4 N NaOH. Detection of telomeric DNA fragments was performed as described elsewhere (2). Nondenaturing in-gel hybridization was performed as described previously (11).
RESULTS
Isolation of the cdc13-4 mutant.Yku−mutant cells are temperature sensitive for growth (14, 15). To investigate the essential role of the Yku heterodimer at 37°C, we performed a synthetic lethality screen to isolate mutants in which YKU70 would be essential for viability. Therefore we disrupted the YKU70 gene in K2348α and tested the resulting mutant KαL7 for phenotypes specific for Yku−mutants. KαL7 is temperature sensitive for growth at 37°C, deficient in nonhomologous end joining, slightly sensitive to methyl methanesulfonate, and displays shortened telomeres (data not shown). The YKU70 gene cloned into plasmid pCH1122 (pCH-YKU70) complemented the phenotypes of KαL7, indicating a functional expression of YKU70 from the plasmid. KαL7-pCH-YKU70 colonies grown on YPD displayed a red-white sectoring phenotype, showing that pCH-YKU70 was not essential for growth at 30°C under nonselective conditions.
After EMS mutagenesis of KαL7-pCH-YKU70 we isolated five stably red mutants, which clearly required YKU70 expression for viability (see Materials and Methods). To identify the mutated gene causing the requirement for YKU70 expression we transformed one mutant, LDM29, with a single-copy yeast library and screened for sectoring colonies indicating that pCH-YKU70 was no longer essential for viability. Plasmids isolated from 15 sectoring colonies revealed three independent clones, two of them carrying a DNA fragment containing the full-length YKU70 gene. The third plasmid, GP2a, contained a fragment of chromosome IV from YDL57269 to YDL68607. This fragment encoded five ORFs, among them YDL220c coding forCDC13/EST4.
Cdc13p, like the Yku heterodimer, has been shown to be an important factor for telomere maintenance. Therefore we subcloned theCDC13 gene from plasmid GP2a into the single-copy vector pRS314 (51). After transformation with the resulting plasmid pRS314-CDC13, LDM29 displayed a clear sectoring phenotype indicating that a mutation in CDC13 caused dependence on Yku70p expression (data not shown). Using the gap repair method (42) we identified a 773-bp fragment near the 5′ end of the CDC13 gene carrying the mutation. Sequencing of this fragment revealed the presence of a single point mutation (at position 703, changing a cytosine to a thymine), thereby leading to the amino acid exchange proline 235 to serine (P235S). Since this mutation differs from the CDC13 mutants already described in the literature, we designated it cdc13-4.
Synthetic lethality of cdc13-4 with Yku.We isolated the cdc13-4 mutation in a synthetic lethality screen with YKU70. To verify the synthetic lethal phenotype we reintroduced the cdc13-4 mutation in the homozygousyku70 strain WaLαU. Therefore we disrupted theCDC13 gene in WaUαL and transformed the resulting strain LDY46, heterozygous for CDC13, with pRS-cdc13-4. As expected, we obtained only two colony-forming spores after sporulation and tetrad dissection of LDY46-pRS-cdc13-4 (data not shown). None of the viable spores was resistant to G418 (the KanMX marker gene was used for CDC13 disruption), indicating that all viable spores contain the wild-type allele of CDC13. The nonviable spores were examined by microscopy. We found many of these spores germinated but arrested at a two-cell stage. In a very few cases we observed microcolonies containing up to 20 cells, which lysed after 2 to 3 days of incubation at 30°C. To show that these phenotypes were not due to synthetic effects caused by the RAD5 mutation in the W303 background (13), we repeated the experiment in a CEN.PK2 strain. In this case we examined the synthetic lethality of cdc13-4 in a yku70- and ayku80-deficient CEN.PK2 strain, LDY54 and LDY55, respectively.
Again we found only two colony-forming spores for most of t