Strain Construction. All strains in this study were isogenic with W303a (a ade2-1 his3-11, 15 ura3-1 leu2-3, 112 trp1-1 rad5-535; ref. 1) except for changes introduced by transformation or by crosses with isogenic strains. Details of strain construction are in Table 3. Sequences of oligonucleotides used in the constructions are in Table 4.

Haploid strains with lig4, yku80, ddc1, mec3, and pds1 deletions were generated by using the PCR-based procedure of Wach et al. (6) to replace the coding sequences with the KANMX cassette. The names of the primers used for these deletions (sequences in Table 4) were: LIG4-UP and LIG4-LW, YKU80-UP and YKU80-LW, DDC1-del-F, and DDC1-del-R, MEC3-del-F, and MEC3-del-R, and PDS1-del-F and PDS1-del-R. The substrate for the PCR reactions was the plasmid pFA6a-kanMX (7) DNA. The fragment generated by PCR was transformed into various haploid strains, as indicated in Table 3, selecting for geneticin-resistant derivatives. All constructions were confirmed by PCR.

The haploid strain MAB1 was constructed in two steps. First, RCY317-9a (an α RAD5 derivative of W303a; ref. 4) was transformed with a PCR fragment generated by amplifying the plasmid pAG32 (which contains a gene conferring resistance to hygromycin; ref. 8) with the primers CHRV F and CHRV R. The resulting strain (PG128.1) had an insertion of the HYG gene between bases 9229 and 9230 of chromosome V; this allele is V9229::HYG. Strain PG128.1 was transformed by a PCR fragment generated by amplifying yeast genomic DNA with primers PG/HIS3F and PG/HIS3R. The resulting strain (MAB1) has an insertion of HIS3 between bases 261553 and 261554 of chromosome V; this allele is V261553::HIS3.

The crosses used to make diploids (haploid strains shown in parentheses) were: JMY309 (JMY303-4b × JMY303-8a; constructed by J. Mallory), KRY237 (Y604 × KRY229 spore [tlc1::LEU2 genotype]; constructed by K. Ritchie), PM183 (PM166(3) × PM198-3d), PM195 (PM194 × PM197-3c), PM197 (SPY41 × RCY308-7b), PM198 (KRY277-2c × RCY308-7b), PM199 (PM198-6a × PM183-3d), PM203 (PM195-1c × PM183-6b), PM204 (KRY237-9d × PM183-2c), MD269 (PM183-3c × MAB1), and MD275 (MD269-14a × PM204-9a). The diploid PM184 was constructed by mating PM198-13d and PM198-10a and transforming the resulting strain with the plasmid pVL1107. This LEU2- and CEN-containing plasmid encodes a fusion protein with the DNA-binding domain of Cdc13p fused to Est2p, the catalytic protein subunit of telomerase (9). The diploids JMY303 (2), KRY229 (2), and KRY277 (3) have been described previously.

We also constructed diploids heterozygous for genes encoding epitope-tagged versions of Xrs2p (PM115), Yku80p (PM116), or Mre11p (PM121). These tags (13× Myc with a KANMX gene located downstream of the tagged protein) were introduced at the C termini of the Xrs2, Yku80, and Mre11 proteins by using the PCR-based method described by Longtine et al. (7). The pairs of primers used for these constructions were: XRS-F2 and XRS2-R1, YKU80-F2 and YKU80-R1, and MRE11-F2 and MRE11-R1 (Table 4). These pairs of primers were used to amplify pFA6a-13Myc-kanMX (7), and the resulting fragments were then transformed into the diploid JMY309. The resulting strains were heterozygous for the alleles XRS2-Myc13-KANMX (PM115), YKU80-Myc13-KANMX (PM116), or MRE11-Myc13-KANMX (PM121). We confirmed the correct transplacements in spores derived from these diploids by PCR.

Strains Used in Generating the PCR Data Shown in Table 1. Most of these strains were derived by sporulating heterozygous diploids (see Table 3). The names of the diploids from which the spores were derived for each genotype are: wild-type (JMY309, KRY237, PM183, PM197, PM199); tel1 (JMY309, PM183, PM197, PM198); mec1 (JMY309, KRY237, PM183, PM197, PM198); mec1 tel1 (JM309, PM183, PM195, PM197, PM198); mec1 tel1 +pVL1107 (PM184); mec1 mre11 (PM198); mec1 tel1 mre11 (PM198, PM199); tel1 mre11 (PM198); tlc1 (KRY237); mec1 tlc1 (KRY237); yku70 (PM197); yku80 (PM195); mec1 yku70 (PM197); mec1 yku80 (PM195, PM203); tel1 yku70 (PM197); tel1 yku80 (PM195, PM203); mec1 tel1 yku70 (PM197); mec1 tel1 yku80 (PM195, PM203); mec1 tel1 lig4 (PM183); and mec1 mre11 lig4 (PM199). Three strains were haploids constructed by transformation: mec3 tel1 (PM206), ddc1 tel1 (PM207), and pds1 tel1 (PM208).

Yeast DNA Isolation Procedure and PCR Conditions for Detection of Telomere–Telomere Fusions (T-TFs). The primers used to detect T-TFs were ChrXV-UP (5' -AAGAATTCTATGGTTAAATGGGGCAGGGTAACG) and ChrV-30 (5' -AAGAATTCGGTAAGAGACAACAGGGCTTGGAGG). To control for the efficiency of the PCR reaction in different DNA samples, we used primers (HIS4-UP and HIS4-DN) that amplified nontelomeric sequences located within the coding sequence of HIS4. The substrates for the PCR reactions were genomic yeast DNA samples (10). DNA samples were treated with the Sau3A endonuclease. Compared to untreated DNA samples, Sau3A treatment resulted in increased levels of the PCR product diagnostic of the fusion, possibly because the longer DNA fragments in the untreated samples resulted in more extensive nonproductive DNA synthesis. For the PCR reactions, we used the Thermal Ace Polymerase Kit (Invitrogen) with the reaction mixtures specified by the kit. The volume for each sample was 30 μl. PCR reactions were performed with the GeneAmp PCR System 9700, ramping 100%. To detect the T-TF product, we amplified the digested DNA by using high-stringency conditions (denaturation at 98° C for 3 min followed by 35 cycles at the following temperatures and times: 98° C, 30 sec; 57° C, 30 sec; 72° C, 2 min). After these cycles, the samples were incubated at 72° C for 7 min. For the control PCR reactions with the HIS4-specific primers, we used 20-25 cycles. The PCR products were separated on a 1.5 % agarose gel and visualized by ethidium bromide staining. For sequence analysis, T-TF PCR products were extracted from the agarose gel by using the QIAquick gel-extraction kit (Qiagen, Chatsworth, CA). The resulting fragments were treated with EcoRI and inserted into the EcoRI site of the pUC19 vector.

As discussed in the text, one potential artifact in the analysis of T-TFs by PCR is that fusions involving long telomeric repeats might be more difficult to amplify than those involving short repeats. We examined this issue by in vitro construction of a fusion involving telomeres of wild-type (350 bp) length by using the plasmid pYLPV (11). A KpnI fragment from this plasmid has 350 bp of telomeric sequence at each end, in the same orientation that would be present at the ends of a chromosome. We self-ligated this fragment, creating a T-TF between telomeres of wild-type length. Dilute solutions of this fragment were then PCR amplified with two primers, one oriented toward one telomere-containing end and one oriented toward the other. The PCR primers used in this experiment were YIp5-tel-test and ARS-LW1 (Table 4). After PCR, we found a fragment of the expected size (≈850 bp) with restriction enzyme sites expected for the fusion (data not shown). We conclude that our PCR method is capable of detecting fusions of wild-type length telomeres and, therefore, the lack of PCR products characteristic of such fusions in certain genotypes indicates that they do not exist or that they are present at low levels.

Real-time PCR was performed by using the same primers described above for the semiquantitative method; all samples were examined by both the T-TF-specific primers and the control primers. The accumulation of double-stranded DNA was detected by using the intercalating dye SYBR green with the Applied Biosystems PRISM 7700 Sequence Detection System. Reactions (50 μl volume) contained Thermal Ace Buffer (Invitrogen), 200 μM dNTPs, 30 pM of each primer, two units of Thermal Ace Polymerase (Invitrogen), and 500 ng of Sau3A-treated genomic DNA. The reactions also contained a 1.5 × 10⁵-fold dilution of the SYBR dye (10,000× concentration provided by Molecular Probes) and the manufacturer-recommended concentration of the fluorescent dye Rox (Invitrogen); the Rox dye is used to establish a baseline for the PCR measurements. The PCR reaction was started by incubating the reaction for 3 min at 98° C. We then did 40 cycles of 94° C for 30 sec, 57° C for 30 sec, and 72° C for 2.5 min, followed by a fluorescence measurement. After 40 cycles, the products of the reactions were examined by gel electrophoresis. Although most (80%) of the samples contained only PCR fragments representing T-TFs, several of the samples had a fragment representing a primer-dimer. For those samples, the amount of this fragment was quantitated (IMAGEQUANT, Molecular Dynamics) and subtracted for the calculations described below.

To calculate the frequency of T-TFs per genome, we determined the difference in the number of PCR cycles required to produce the same amounts of the control HIS4 PCR fragment (present in one copy/genome) and the T-TF-specific PCR fragment. This calculation was done by using the threshold values set by the Applied Biosystems PRISM software (≈40% of the final level of the DNA fragments) and setting the baseline to the average of cycles 3-10. The frequency of T-TFs per genome was calculated with the formula: T-TFs/genome = 1/(2^N), with N representing the difference in the number of cycles for the HIS4- and T-TF-specific PCR fragments. Ninety-five percent confidence limits for the N values were calculated by using INSTAT 1.12 (GraphPad, San Diego). This calculation assumes that the control and TTF PCR fragments are amplified with equal facility. This assumption is supported by two findings. First, the curves representing the increasing levels of the two DNA fragments as a function of the number of cycles are approximately parallel. Second, when we mixed an amount of a purified T-TF fragment equivalent to one copy per genome with wild-type yeast genome DNA and performed the PCR analysis, we found that the number of cycles required to detect the T-TF fragment was about the same (within one cycle) as that required to detect the control HIS4 fragment.

Chromatin Immunoprecipitation Assay (ChIP). The methods for the ChIP were as described by Burke et al. (10), except that for each immunoprecipitation, we used 15 mg of protein extract, and the immunoprecipitates were washed with a 1 M NaCl buffer. For the Rap1p experiments, we used polyclonal goat antibody to Rap1p (Santa Cruz Biotechnology) diluted 1:750, and Protein A/G PlUS-Agarose (Santa Cruz Biotechnology). As a negative control, we used normal goat IgG diluted 1:750. The Myc-tagged proteins (Yku80, Mre11, and Xrs2) were precipitated with monoclonal mouse anti-Myc antibody (Babco, Richmond, CA) (diluted 1:750) and protein A/G PlUS-agarose; normal anti-mouse IgG (Santa Cruz Biotechnology) (diluted 1:750) was used as a control. The following primers were used for multiplex PCR (the first two to amplify DNA near the telomere, the second two to amplify the ACT1 gene): CXV202UP and CXV469LW, and ACT1IP-UP1 and ACT1IP-LW1. DNA was amplified (by using ³²P-labeled dCTP, in addition to unlabeled nucleotides) under high stringency conditions (denaturation 94° C, 3 min; 22 cycles, 94° C, 30 sec; 59° C, 30 sec; 72° C, 1 min; 72° C, 7 min). PCR products were separated on a 6.6% polyacrylamide gel and visualized by the PhosphorImager system (Molecular Dynamics).

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