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Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324,1 Department of Cell and Molecular Biology, Lundberg Laboratory, Göteborg University, S-405 30 Göteborg, Sweden2
Received 25 July 2002/ Returned for modification 9 September 2002/ Accepted 9 February 2003
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ABSTRACT |
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, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a pol mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Pol was severely compromised compared to those in the wild-type pol cells. Interestingly, ectopic expression of wild-type pol in this pol mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Telomeres are also essential for the complete replication of eukaryotic chromosomes. DNA synthesis by DNA polymerases has a 5'-to-3' polarity (38). At the replication fork, the leading-strand synthesis is thought to initiate once and then proceeds continuously. The lagging-strand synthesis is discontinuous throughout and requires repeated initiations by polymerase (Pol) and primase (9, 12, 73). Due to the polarity of DNA synthesis, the telomeric G-rich strand is synthesized by the leading-strand replication machinery, and the C-rich strand is synthesized by the lagging-strand replication machinery. Removal of the terminal RNA primer by nucleases leaves an 8- to 12-nucleotide gap at the 5' end of the newly replicated DNA that cannot be refilled by conventional DNA replication, resulting in an end replication dilemma (44, 74). The end replication problem is potentially resolved by telomerase, an unusual reverse transcriptase. The catalytic subunit of telomerase (Trt1) contains an integral RNA molecule with a small template domain that is utilized by telomerase as template to add telomeric repeats onto the 3' end of the telomere (57). Although telomerase activity is required to maintain the steady-state length of telomeric DNA repeats at the chromosome end, other proteins have been shown to affect telomere length maintenance. In Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast), proteins that bind either directly to telomeric sequences or to telomeric sequence-binding proteins have been shown to positively or negatively influence telomere length (10, 11, 27, 43, 57). In addition, mutations in several checkpoint genes in fission or budding yeast and in Caenorhabditis elegans have been shown to affect telomere length (3, 22, 42, 48, 51, 54, 66).
Mutations of components of budding yeast DNA replication machinery such as POL1 (Pol) or the large subunit of replication factor C (CDC44/RFC1) confer telomere elongation, whereas a rad27 deletion confers destabilized telomere length (1, 15, 60). Moreover, in a POL1 mutant (pol1-17), the telomeric position effect (TPE) is reduced concomitantly with telomere elongation (2). Recent data have shown that fission yeast Pol mediates recruitment of Swi6 to heterochromatin, including that of telomeres to establish silencing (4). By using a novel in vivo assay, it has been shown that telomerase-mediated telomere addition requires the activities of Pol, Pol
, and primase in budding yeast (23). In budding yeast, Cdc13p plays an important role in protecting and maintaining telomeric DNA (31, 56). Budding yeast Cdc13p interacts with the catalytic subunit of DNA Pol (Pol1p) by two-hybrid analysis criteria. Point mutations in either CDC13 or POL1 not only reduce the interaction but also affect telomere length (64). Aphidicolin, an inhibitor of Pol and Pol, causes abnormal lengthening of the G- and C-strand heterogeneity of telomeres of the ciliate Euplotes (28), and Euplotes telomerase has been shown to physically associate with primase, a component of the lagging-strand machinery (65). Together, these studies suggest that telomere replication by telomerase requires coordination of C- and G-strand syntheses.
Fission yeast is evolutionarily distant from budding yeast. The chromosome organizations of fission yeast, such as centromere structure (18, 19), the replication origin structure (24), and chromosome segregation (5), are more similar to those of mammalian cells than are those of budding yeast. Much less is known about fission yeast telomeres than about their budding yeast counterparts. Fission yeast telomeres consist of about 300 bp of repeat units, which includes a consensus sequence of 5'-TTACAG1-8-3' on the strand that runs towards the 3' end (25, 68). To investigate whether telomere length maintenance in fission yeast is similar to that in the evolutionarily distant budding yeast, we have analyzed the telomere length of a large panel of replication mutants. We report here that telomere length is altered in all of the replication mutants analyzed. Using the principal initiation and lagging-strand synthesis DNA polymerase (Pol) as a representative of replication proteins, we show here that Pol and the telomerase catalytic subunit, Trt1, coexist in a complex in vivo. Analysis of the telomere length and the Trt1 protein level in a pol mutant reveals a close physical association between the replication complex and the telomerase complex in S. pombe. Mutations in components of the replication complex, such as Pol, could affect the telomeric complex in maintaining telomere length and telomerase protein stability.
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MATERIALS AND METHODS |
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double mutant strain, the CF248 diploid strain (h+/h- leu1-32/leu1-32 ura4-D18/ura4-D18 his3-D1/his3-D1 ade6-M210/ade6-M216 trt1+/trt1::his3+) was sporulated, and the spores were germinated on Edinburgh minimal medium (EMM) (50) lacking histidine to select for trt1::his3+. This strain was crossed with an h- spp2-9 strain (69), which had grown for 100 generations, to generate the spp2-9 trt1 mutant. The spp2-9 trt1 double mutant was verified by temperature sensitivity and its ability to grow on media lacking uracil and histidine. Tetrads were dissected and germinated on YES (yeast extract plus supplements). The polts13 strain [pKAN1-C-myc9trt1+ (kanMX Cmyc9trt1+)] (hereafter the polts13 myc-trt1+ strain) was made by crossing the h+ polts13 strain (13), which had been grown for 100 generations, and the CF830 (h- leu1-32 ade6-M210 ura4-D18 his3-D1 trt1::his3+ [pKAN1-C-myc9trt1+ (kanMX Cmyc9trt1+)] strain (36). The polts13 myc-trt1+ mutant strain was verified by Western blotting with anti-myc antibody, temperature sensitivity, and Southern blot analysis of telomere length. The polts13 ura-tel strain was constructed by crossing the h- polts13 strain, which had been grown for 100 generations and the FY1872 strain (h90 leu1-32 ade6-210 his3D1 ura4DS/E ade6OTRsph ura4-tel) and was verified by PCR with primers 5'-TGAGGGGATGAAAAATCCCATTG-3' and 5'-TTCGACAACAGGATTAC-GACCAG-3' directed against the ura4+gene. The polts13 strain [pKAN1-Cmyc9trt1+ (kanMX Cmyc9trt1+)] [pART-pol+ (ura4+ pol+)] (hereafter the polts13 myc-trt1+ + pol+ strain) was constructed by transforming the polts13 myc-trt1+ strain with pART-pol+, followed by selection for expression of pol+ by shifting the temperature to 36°C to rescue the temperature sensitivity of the polts13 myc-trt1+ strain. The CF830 (pol+ myc-trt1+), polts13 myc-trt1+, and polts13 myc-trt1+ + pol+ strains were grown in yeast extract with G418 (0.1 mg/ml) at 25°C to maintain the episomal plasmid pKAN1-C-myc9trt1+; all other strains were grown in YES at the indicated temperatures. Detection of telomeres by Southern blotting. Genomic DNA was isolated by a glass bead-phenol protocol and analyzed on agarose gels. Agarose gels were stained with ethidium bromide (EtBr) and photographed to ensure that equal amounts of DNA were loaded. A 2.4-kb ApaI fragment of unique S. pombe genomic DNA (22) was also used as a loading control. Detection of telomeres by Southern blotting was carried out as described previously (22).
G-strand overhang assay.
To detect G-strand overhangs, a nondenaturing hybridization assay was used (72). Oligonucleotide probes 5'-GGGTTACAGGTTACAGGGTTAC-3' (G-specific oligonucleotide) and 5'-GTAACCCTGTAACCTGTAACCC-3' (C-specific oligonucleotide) were designed based on reference 20. The C- and G-strand-specific probes were end labeled with [
-32P]ATP (3,000 Ci/mmol; Amersham) by T4 polynucleotide kinase to identical specific activities (9,160 cpm/pmol for the C-strand probe and 9,220 cpm/pmol for the G-strand probe). Labeled probes were purified on MicroSpin G-25 columns (Amersham Pharmacia Biotech). Four microliters of labeled probe (8 nM) was used for each reaction to hybridize to 5 µg of ApaI-restricted genomic DNA (from cells grown for 100 generations). The labeled probes were added to a final volume of 25 µl and then incubated for 12 to 15 h at 50°C. Hybridized samples were separated on a 1.5% agarose gel, stained with EtBr to ensure equal loading, dried, and autoradiographed.
Preparation of protein extracts from S. pombe. A total of 3 x 108 cells were harvested, washed once in ice-cold stop buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA [pH 8.0], 1 mM NaN3), and then resuspended in 25 µl of lysis buffer (150 mM HEPES [pH 7.9], 300 mM KCl, 1 mM EDTA, 10% glycerol), with the addition of 1 tablet of Roche Complete Protease Inhibitor Cocktail per 25 ml of buffer, and lysed by vortexing with glass beads. Soluble protein extracts were prepared by centrifugation at 4°C for 10 min, and the protein concentration was determined by the Bradford assay.
Immunoprecipitation and Western blotting.
Five hundred micrograms to 1 mg of total protein was solubilzed in 1 ml of lysis buffer containing 0.2% RNase inhibitor RNasin (Promega) and 0.1% NP-40. Anti-c-myc (9E10) antibody was preadsorbed onto protein G plus/protein A agarose beads (Oncogene Research). Affinity-purified anti-Pol antibody (61) was conjugated with CNBr-activated Sepharose beads according to the manufacturer's instructions. Ten microliters of a 50% slurry of antibody-labeled beads was added to the protein extract, and this mixture was incubated for 2 to 3 h at 4°C with end-over-end rotation. Immunocomplexes were washed four times with lysis buffer and resuspended in 30 µl of sodium dodecyl sulfate (SDS)-sample buffer. Ten microliters was analyzed on SDS-polyacrylamide gel electrophoresis (PAGE) gels (8% polyacrylamide) and then transferred to polyvinylidene difluoride membranes. The membrane was blocked in 5% dry milk powder in phosphate-buffered saline (PBS) and 0.2% Tween 20. Anti-Pol antibody at a 1:4,000 dilution, anti-myc antibody at a 1:500 dilution, and antitubulin antibody at a 1:5,000 dilution were used as the primary antibodies. A rabbit anti-chicken secondary antibody at a 1:1,000 dilution was used for detecting anti-Pol immunoglobulin Y (IgY), and a goat anti-mouse secondary antibody at a 1:3,000 dilution was used for detecting anti-myc. Proteins were detected by the ECL enhanced chemiluminescence system (New England Nuclear). Five milligrams of EtBr per ml was added to the myc-trt1 cell extracts prior to immunoprecipitation.
Cell synchronization. The myc-trt1+ strain was grown at 25°C to a cell density of 6 x 106 cells per ml before hydroxyurea (HU) was added to a final concentration of 12 mM. The cells were incubated in HU for 4 h and then released into media without HU. Cell samples of 50 ml were taken every 15 min. One milliliter of the cell sample was fixed with ethanol. A total of 3 x 106 fixed cells were stained with propidium iodide for flow cytometry analysis (62). The rest of the cells (49 ml) were used for protein extract preparation.
Telomere position effect.
The pol+ (ura4-tel) (FY1872), polts13 (ura4-tel), and polts13 (ura4-) strains were grown to 5 x 106 cells per ml at 25°C. Serial dilutions were applied as spots to PM + adenine-histidine-leucine-uracil (AHLU) (complete), PM + AHL (ura-), and PM + AHLU + 5-fluoroorotic acid (5-FOA) plates and grown at 25°C for 4 days or until colonies appeared.
Ectopic expression of pol+.
Full-length pol+ was expressed from pART-pol+ in the polts13 myc-trt1+ strain. The myc-trt1+ (CF830), polts13 myc-trt1, and polts13 myc-trt1+ + pol+strains were grown at 25°C to 6 x 106 cells per ml, and the cultures were then divided into two halves: one-half was shifted to 36°C to select for cells sustained by pART-pol+, while the other half continued culturing at 25°C. Cell samples were taken at 6 and 15 h after shifting the temperature to 36°C for protein extract and DNA preparation. Western blotting and telomere length analyses were performed as described above.
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RESULTS |
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100 generations at its respective semipermissive temperature in liquid media. Many of the mutant strains analyzed in the experiments had been propagated for many generations prior to this study, and consequently they had already reached their steady-state telomere lengths before 100 generations of subculture. We chose to routinely analyze strains after 100 generations of subculture and compared them to their respective isogenic parental strains. We first analyzed mutants with mutation of primase and pol that are essential for initiation of Okazaki fragments. All five mutants with mutation of the primase catalytic subunit, spp1 (33), exhibited an average of 400- to 600-bp extensions of telomeres compared to their parental wild-type strains (Fig. 1A, compare lanes 2 to 6 to lane 1). Out of these spp1 mutants, the spp1-9 mutant had the longest telomeres (Fig. 1A, lane 3). The two pol mutants (13) displayed differences in their telomere length after subculture for 100 generations at 25°C. The polts11 mutant had telomeres about 270 bp longer than those of the wild type (Fig. 1A, lane 7), while the polts13 mutant had a majority of telomeres 550 bp longer than those of the wild type (Fig. 1A, compare lanes 1 and 8). Longer exposure of the gel revealed that a minor population of the telomeres in the polts13 strain were 1,600 bp longer than wild-type telomeres (appearing as a weak smear in Fig. 1A, lane 8). Of the three spp2 mutants, the mutant containing the gene encoding the primase coupling subunit, spp2-9 (69), exhibited the longest telomeres: 300 bp longer than those of the wild type (Fig. 1B, lane 4). These results show that the extent of telomere lengthening in primase and pol mutants is allele dependent.
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mutants (30, 58). All five pol mutants had on average 100- to 150-bp-longer telomeres than their respective isogenic wild-type strains (Fig. 2A, lanes 3 to 7). Furthermore, mutants with defects in two small subunits of pol, cdc1 and cdc27 (46), exhibited telomere lengths about 180 bp longer than those of the wild type (Fig. 2B, lanes 2 and 3). Similar to pol and pol mutants, a mutant with a ligase mutation, cdc17-K42, had telomeres about 160 bp longer than those of the wild-type strain (Fig. 2B, lane 4). Although the exact role of Pol
in replication is not yet clear, similar to a recent observation in S. cerevisiae (59), a pol mutant, the cdc20-M10 strain, exhibited shorter telomeres than the wild-type strain (Fig. 2B, lane 5). A strain with deletion of rad2, encoding a nuclease required for Okazaki fragment maturation (73), also had shorter telomeres than those of the wild-type strain (Fig. 2B, lane 6). Thus, mutations of genes encoding fission yeast replication proteins induce deregulation of telomere length.
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and polts13 mutants exhibited substantially longer telomeres than the wild type, we further analyzed whether the telomeric extension in these mutant strains is a G- or C-strand extension as described in Materials and Methods. With C- or G-strand-specific probes of identical radioactive specific activities, the results suggest that the telomere elongation seen in these replication mutants is an extension of the G strand (data not shown).
Mutation at a pol mutant allele, polts13, affects the silencing at telomere loci.
It is known that mutation of genes that causes a telomere length phenotype could also affect the TPE. In S. cerevisiae, the pol1-17 allele causes elongation of telomeres and reduces the TPE (2). We therefore investigated the possible effect of the polts13, spp1-9, and spp2-9 mutations on silencing at the telomere locus. The polts13 ura4-tel strain with the ura4+ gene placed at a telomere locus was constructed as described in Materials and Methods. The polts13 ura4-tel mutant was able to grow on media lacking uracil, indicating a slight reduction of silencing at the telomere locus (Fig. 3). This result corroborates a recent report that showed an interaction between Pol and Swi6, an element required for silencing. In a thermosensitive pol mutant (the swi7-H4 strain), the interaction was impaired and the silencing was defective (4). In contrast, the spp1-9 and spp2-9 mutations do not affect the TPE (data not shown).
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and primase mutants is telomerase dependent.
We then investigated whether the telomere length extension occurring in these replication mutants is mediated by telomerase. Attempts were made to generate polts13 (13) or primase subunit spp1-9 (33) and spp2-9 (69) double mutants in the telomerase catalytic subunit (trt1) deletion background (16, 52, 53) as described in Materials and Methods. However, both random sporulation and crossing followed by tetrad analysis failed to generate spp1-9 trt1 or polts13 trt1 double mutants. Mating of the trt1 strain with either the spp1-9 or polts13 mutant strain resulted in one to two spores per tetrad, with none of them being the desired double mutant. Hence, both the spp1-9 and polts13 strains have a strong synthetic interaction with deletion of trt1+. In contrast, the spp2-9 trt1 double mutant was successfully generated. The mutant spp2-9 trt1 double mutant was cultured in liquid medium at the semipermissive temperature (33°C) up to 100 generations, and its telomere length was compared to the telomere lengths of the wild type and the spp2-9 single mutant. The telomere length of the spp2-9 single mutant after subculture for 100 generations was about 300 bp longer than that of the wild type (Fig. 4, compare lane 2 to lane 1). After growing for 30, 50, 70, and 100 generations, the spp2-9 trt1 mutant exhibited progressively shorter telomere lengths (Fig. 4, lanes 3 through 6). After 100 generations of growth, the telomere length was even shorter than that of the parental wild-type strain (Fig. 4, compare lane 6 to lanes 1 and 7). This result strongly suggests that the telomere lengthening seen in the spp2-9 mutant requires the action of telomerase. By extending the interpretation of our results, the telomere lengthening in the pol and primase mutants may be also mediated by telomerase.
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and Trt1 physically interact in vivo.
Studies of S. cerevisiae and Euplotes have suggested a coordination of replication of the two strands at the telomere (23, 28, 47, 65). Our results indicate that Trt1 is responsible for the aberrant telomere extension in the pol and primase mutants (Fig. 4). Furthermore, polts13 and trt1 strains showed a strong synthetic phenotype. These data bring to mind a physical interaction between Pol and telomerase. To test this possibility, coimmunoprecipitation experiments were performed with a myc-tagged trt1+ strain ectopically expressed at a moderate level from its own promoter in a trt1 background (CF830) (36), and a wild-type nontagged trt1 strain as a negative control. An 130-kDa myc-tagged Trt1 protein, but no protein of the size of Pol, was detected when cell extracts prepared from the myc-trt1+ strain were probed with the anti-myc monoclonal antibody 9E10 (Fig. 5A, lane 1). When the same cell extract was probed with polyclonal anti-Pol antibody (61), three protein species of Pol were detected—a major band of 180 kDa and minor amounts of 165 and 155 kDa, which are degraded forms of Pol (61): however, no protein band of the same size as myc-tagged Trt1 was detected (Fig. 5A, lane 2). These results indicate that the anti-myc and anti-Pol antibodies do not cross-react with Pol and myc-Trt1, respectively.
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with anti-Pol from myc-trt1+ cell extracts and probing with anti-myc antibodies, a myc-Trt1 protein was detected (Fig. 5B, lane 1). As a control, myc-Trt1 was detected from the same cell extracts when immunoprecipitated and probed with anti-myc antibodies (Fig. 5B, lane 2). No myc-Trt1 was detected in immunoprecipitates made from the nontagged wild-type strain (Fig. 5B, lane 3 and 4). Reciprocally, Pol was detected in the anti-myc immunoprecipitates from the myc-trt1+ strain when probed with anti-Pol antibody (Fig. 5B, lane 6). To verify that this was indeed Pol, the anti-Pol immunoprecipitate from the same cell extract was probed with anti-Pol antibody. It exhibited proteins of the size of Pol (Fig. 5B, lane 5). Anti-Pol-antibodies detected Pol in anti-Pol immunoprecipitates from lysates of the nontagged wild-type trt1 strain (Fig. 5B, lane 7), whereas no Pol could be detected in the anti-myc immunoprecipitates from the same cell extract (Fig. 5B, lane 8). Thus, Trt1 and Pol can be reciprocally coimmunoprecipitated by these antibodies.
To ascertain that the coimmunoprecipitation of Trt1 and Pol was not caused by a random nonspecific coassociation with DNA or RNA fragments in the cell extract, EtBr was added to the myc-trt1+ cell extract prior to immunoprecipitation. myc-Trt1 was detected in the anti-Pol-immunoprecipitates from cell extracts with or without EtBr added (Fig. 5C, lanes 2 and 4). Thus, the coimmunoprecipitation of Trt1 and Pol was not due to nonspecific random association of these proteins with nucleic acids in the cell extract. Together, these results strongly suggest that telomerase and DNA Pol physically interact in vivo.
We then investigated whether the association of Trt1 and Pol is cell cycle dependent. The myc-trt1+ strain was arrested in early S phase with HU at 25°C for 4 h and then released into fresh medium to continue growing at 25°C. After release from the HU arrest, the cells took approximately 60 min to recover and progress through S phase. Cell samples were removed at 0, 60, 75, and 120 min after release from the HU block for flow cytometry analysis (Fig. 6A) and for preparation of protein extracts for coimmunoprecipitation experiments (Fig. 6B and C). Cells arrested in HU for 4 h at 25°C displayed a 1C DNA profile (Fig. 6A, t = 0 min). The cells gradually shifted towards a greater-than-1C fluorescence-activated cell sorter (FACS) profile, and after 120 min, cells had a 2C DNA profile, indicating that cells had completed S phase and entered the G2 phase (t = 120 min). Since S. pombe has a very abbreviated G1 phase, no G1-phase cells are observed in the 120-min cell sample. Throughout S phase and in G2 phase, myc-Trt1 was detectable in the anti-Pol immunoprecipitates (Fig. 6B, lane 2) and the anti-myc immunoprecipitates (Fig. 6B, lane 3). Pol was effectively detected by the anti-Pol antibody in the anti-Pol immunoprecipitate (Fig. 6C, lane 2). Furthermore, Pol coimmunoprecipitated with myc-Trt1 as cells progressed through S phase and entered the G2 phase, although the level was low at t = 0 min (Fig. 6C, lane 3, t = 0, 60, 75, and 120 min). These experiments indicate that Trt1 and Pol interact throughout S and G2 phases of the cell cycle.
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and Trt1 is compromised in a pol mutant exhibiting long telomeres.
To test the status of interaction between Trt1 and Pol in a pol mutant (the polts13 strain) that exhibited abnormally long telomeres (Fig. 1A, lane 8), we constructed a strain containing the polts13 allele and myc-trt1+ and analyzed the interaction of these two proteins. As expected, myc-Trt1 was detected in anti-myc immunoprecipitates (Fig. 7A, lane 1) and also coimmunoprecipitated with Pol (Fig. 7A, lane 2), and Pol was detected in anti-Pol immunoprecipitates from cell lysates of the myc-trt1+ (pol+) strain (Fig. 7A, lane 3). In contrast, although myc-Trt1 was immunoprecipitated by identical amounts of anti-myc antibody from myc-trt1+ (pol+) (Fig. 7A, lane 1) and polts13 myc-trt1+ cell extracts (Fig. 7B, lane 1), no detectable myc-Trt1 was found in the anti-Pol immunoprecipitates from the polts13 myc-trt1+ cell extract (Fig. 7B, lane 2). This is not due to the failure of anti-Pol antibody to immunoprecipitate the mutant Pol protein, because mutant Pol protein was readily detectable in the anti-Pol immunoprecipitates from polts13 myc-trt1+ cell extract (Fig. 7B, lane 3).
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ts13 myc-trt1+ mutant, we tested whether protein levels of Pol or myc-Trt1 were altered in the polts13 myc-trt1+ mutant. Cell extracts of 0.1 to 1 mg of protein from the myc-trt1+ (pol+) and polts13 myc-trt1+ strains were Western blotted to determine the levels of the Pol and myc-Trt1 proteins (Fig. 7C, top two panels). Comparable levels of Pol protein were present in both myc-trt1+ (pol+) cells and the polts13 myc-trt1+ mutant (Fig. 7C, first panel). Surprisingly, the level of myc-Trt1 protein was severely reduced in the polts13 myc-trt1+ mutant (Fig. 7C, second panel). The level of myc-Trt1 in polts13 myc-trt1+ mutant cell extract was about 10% of the myc-Trt1 level in the myc-trt1+ (pol+) cell extract: compare the myc-Trt1 protein level in 0.1 mg of myc-trt1+ (pol+) cell extract with the myc-Trt1 protein level in 1 mg of polts13 myc-trt1+ cell extract. We then analyzed the interaction of myc-Trt1 and Pol in these cell extracts. With comparable levels of Pol protein immunoprecipitated from myc-trt1+ (pol+) and polts13 myc-trt1+ cells, myc-Trt1 was detectable in the anti-Pol immunoprecipitates from myc-trt1+ (pol+) cell extracts, but not in polts13 myc-trt1+ extracts. Furthermore, with comparable levels of myc-Trt1 presented in 0.1 mg of myc-trt1+ (pol+) extract and in 1 mg of polts13 myc-trt1+ cell extract, myc-Trt1 protein was again detectable only in the anti-Pol-immunoprecipitates from 0.1 mg of myc-trt1+ (pol+) cell extract, but not from 1 mg of polts13 myc-trt1+ cell extract (Fig. 7C, panels 4 and 5). Moreover, although 1 mg of polts13 myc-trt1+ cell extract had a much higher level of Pol protein than 0.1 mg of myc-trt1+ (pol+) cell extract (Fig. 7C, panel 1), myc-Trt1 was only detectable in the anti-Pol-immunoprecipitates from myc-trt1+ (pol+) cell extract and not in those from polts13 myc-trt1+ extract. These results suggest that the polts13 mutation not only causes an abnormal telomere lengthening, but also severely reduces the cellular Trt1 protein level and compromises the ability of Trt1 and Pol to physically coexist as a complex in vivo.
Ectopic expression of pol+ restores myc-Trt1 level and shortens telomeres in the polts13 mutant.
As shown above in Fig. 7C, the myc-Trt1 protein level in the polts13 myc-trt1+ mutant strain was significantly lower than that in the myc-trt1 (pol+) strain. It is possible that the mutant Pol in the polts13 strain might affect the stability of Trt1 in the Pol-Trt1-containing protein complex, causing Trt1 to become unstable. To test this hypothesis, we transformed pART-pol+ into the polts13 myc-trt1+ strain (hereafter the polts13 myc-trt1+ + pol+ strain) to express the wild-type pol gene. The myc-trt1+(pol+), polts13 myc-trt1+, and polts13 myc-trt1+ + pol+ strains were cultured at 25°C to early log phase and then shifted to 36°C for 6 h to select for cells sustained by the ectopic expression of pol+. At 25°C, when there is no selection for pART-pol+, most cells have probably lost the plasmid, and the myc-Trt1 level is comparable to that of polts13 myc-trt1+ strain (Fig. 8A, lane 2 and 3). The polts13 myc-trt1+ strain without the ectopic expression of pol+ is viable at 36°C for 6 h, but then starts to gradually lose viability, whereas the polts13 myc-trt1+ strain transformed with pART-pol+ is viable. After 6 h at 36°C, the myc-Trt1 level is increased in the polts13 myc-trt1+ + pol+ strain compared to that in the polts13 myc-trt1+ strain (Fig. 8A, compare lanes 5 and 6). After 15 h at 36°C, the level of myc-Trt1 in the polts13 myc-trt1+ + pol+ strain is comparable to that of the myc-trt1+ (pol+) strain expressing wild-type Pol (Fig. 8B, compare lanes 1 and 2), with equal amounts of protein extracts analyzed, as shown by the tubulin levels used as the control. These experiments indicate that ectopic expression of pol+ is able to restore the myc-Trt1 level in the polts13 myc-trt1+ strain, suggesting that Pol affects the physical status of Trt1.
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+ also affects the telomere length in the polts13 myc-trt1+ strain, genomic DNA was prepared from the same strains shown in Fig. 8A and B, and the telomere lengths were analyzed by Southern blotting. Interestingly, ectopic expression of a functional pol+ gene rapidly restored the telomere length to near-wild-type length in the polts13 myctrt1+ mutant (Fig. 8C). These results indicate that Pol plays a significant role in maintaining the Trt1 protein level and also the telomere length of cells, supporting the finding of a close physical association between Pol and Trt1 in vivo. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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associates with Trt1 in a complex in vivo and that the status of Pol influences telomere length as well as the cellular level of the Trt1 protein (Fig. 5, 7, and 8). Below, we discuss these findings and their possible effects on the cell biology. Do replication proteins of fission yeast and budding yeast affect telomere length differently? In this study, we analyzed 20 replication mutants for their telomere length (Fig. 1 and 2). Among these mutants analyzed, mutation of ligase (cdc17-K42) induces abnormal telomere lengthening (Fig. 2B, lane 4). In contrast to the fission yeast ligase mutant (cdc17-K42), an S. cerevisiae mutant with a ligase mutation (cdc9) has telomeres of wild-type length (2). In addition, deletion of fission yeast rad2 (S. pombe homolog of Fen1/RAD27), which is essential for maturation of Okazaki fragments (9, 73), induces telomere shortening (Fig. 2B, lane 6). In budding yeast, deletion of RAD27 (the homolog of S. pombe rad2+) causes destabilization of telomeres but not a shortening of telomeres (60). The differences seen in telomere length regulation of ligase and of rad2+/RAD27 mutants between the two yeasts might simply be due to allele specificity. On the other hand, they might reflect a difference in the effect of replication proteins on telomere length regulation between the two yeasts.
In budding yeast, telomerase-mediated telomere addition requires Pol, primase and Pol, but not Pol (23). Although the exact role of Pol in replication is not yet clear (9, 12, 73), mutation of pol (cdc20-M10) in both fission yeast and budding yeast, similar to fission yeast rad2, induced shortening of telomeres (Fig. 2B, lane 5) (59). This suggests that in fission yeast, pol+ and rad2+ regulate telomere length through a different mechanism from that of the pol+, pol+, and ligase genes. Since Pol, primase, and Pol are all thought to be involved in lagging-strand synthesis, a different kind of telomere length regulation seen in the pol mutant may reflect a unique and distinct role of Pol in replication.
How might the replication complex influence the telomere homeostasis?
Studies of telomeres in the ciliate Euplotes (28, 63, 65) and budding yeast (1, 2, 15, 23, 27, 47, 64) have led to the proposal that G-strand extension and C-strand synthesis require a coordinated regulation. Since C-strand synthesis requires the lagging-strand replication proteins, it is reasonable to assume that synthesis of the two telomeric strands also requires a tight coordination of the lagging-strand replication complex and the telomerase-containing telomeric complex (27). Studies of budding yeast have shown that Pol1p (Pol) interacts with Cdc13p by two-hybrid analysis; mutations in an N-terminal region of Pol1p abolish the interaction and result in longer telomeres (64). Cdc13p also interacts with Est1p (64), which also associates with the telomerase RNA (26, 40, 56). This interaction chain implies that Cdc13p might recruit both telomerase and Pol to the telomeres in budding yeast; hence Cdc13p may be a key linchpin in telomere homeostasis.
In this study, we have shown that the Trt1 and Pol proteins coexist in a complex in vivo (Fig. 5), and the physical and/or functional status of Pol significantly affects the telomerase protein stability and telomere length maintenance (Fig. 7 and 8). It is not yet known whether the two proteins directly interact or whether other proteins mediate the interaction. One S. pombe protein, Taz1, has been identified as a telomere binding protein (21, 29). Taz1 is an ortholog of human telomere repeat binding factors (TRFs). Taz1 is neither a structural nor a functional homolog of Cdc13p (20, 39). Another telomere end binding protein, Pot1, was recently identified in fission yeast and humans (10, 11). It is not yet clear whether Pot1 is an ortholog of Cdc13 and whether Pot1 is a linchpin of the interaction between the lagging strand replication complex component Pol and Trt1. Thus, the coordination of G- and C-strand syntheses in S. pombe may be mediated by a yet-to-be-identified protein. It is also possible that the coordination of the synthesis of G and C strands in fission yeast is by a direct interaction between the Pol-containing lagging-strand replication complex and the telomerase-containing telomeric complex.
The polts13 mutant at 25°C has a growth rate similar to that of the wild type; however, polts13 cells exhibit a slight cdc phenotype with a normal nuclear morphology (13), indicating that 25°C is the semipermissive temperature of this mutant. Moreover, at 25°C, the polts13 mutant exhibits an elevated mutation rate (41); hence, at this temperature, Pol is semidysfunctional. Here we showed that at 25°C, the polts13 mutant allele induced an aberrant lengthening of telomeres (Fig. 1), a mild reduction of TPE (Fig. 3), a severely compromised interaction with Trt1 (Fig. 7B), and a significant decrease in the cellular Trt1 protein level (Fig. 7B and C). It is possible that a semidysfunctional Pol adversely affects the coordination between the lagging strand replication complex and the telomerase complex. The impaired physical coordination between these two protein complexes would make Trt1 prone to degradation, resulting in a decrease in the cellular Trt1 level, and a mild reduction in silencing at the telomere loci due to a change of the heterochromatin structure at the telomere. In support of this hypothesis, ectopic expression of wild-type pol at the restrictive temperature can progressively restore the cellular level of Trt1 protein and reduce telomere length to near-wild-type length (Fig. 8). However, the recovery rates of Trt1 protein level and the telomere length induced by ectopic expression of pol+ in the polts13 are not perfectly correlated. This could be due to a delayed telomere effect. Furthermore, the telomere length also seemed to have reached a new steady-state level, almost as short as the wild-type telomere length (Fig. 8C). This telomere-shortening phenomenon was already seen at the first time point after establishment of the pol+ transformants (Fig. 8C, lane 4) and is most likely due to the preselection of pol+ transformants at 36°C. The fact that telomeres do not quite revert to wild-type length could also be due to an altered extension of heterochromatin in the region near the telomere during the previous phase when the telomeres were abnormally long. If this were the case, it would require many generations to restore the chromatin near telomeres to the original state. Nonetheless, these results suggest that the Pol protein plays a significant role in maintaining telomeric complex stability and telomere homeostasis.
There are two paradoxical findings in our studies. First, Pol and Trt1 interact in early S phase (Fig. 6). At this phase of the cell cycle, no telomere replication presumably takes place. Budding yeast telomere addition at the de novo end occurs in M phase, although telomerase activity can be measured in vitro in both G1- and M-phase cells (23). The finding that Pol and Trt1 coimmunoprecipitate at low levels throughout S phase suggests that a fraction of Pol may constitutively associate with telomerase during the entire S phase. This also implies that another essential factor or factors required for the telomerase-mediated telomere addition may be associating with telomerase in a cell cycle-dependent manner (23).
The second paradox is how the polts13 myc-trt1+ mutant, with a significantly reduced level of Trt1 protein, can exhibit lengthened telomeres. A likely explanation is that cells might have excess amounts of potentially active telomerase, which is usually tightly regulated by the coordination between the telomerase complex, the replication complex, and/or perhaps other proteins. In the polts13 mutant, a dysfunctional Pol might perturb the tight coordination of these proteins in regulating the telomerase-mediated telomere addition. Furthermore, a perturbed telomeric protein complex in the polts13 mutant may allow the chromosome ends to be more accessible to extension by low levels of residual telomerase in an aberrant manner (27). Thus, it is possible that the abnormal telomere lengthening observed in the polts13 strain is a consequence of perturbation of the organization of protein complexes at the telomere end.
Mutations of replication proteins not only contribute to telomere dysfunction but also promote genomic instability.
Telomere dysfunction due to the absence of functional telomerase has been shown to increase mutation rate, impair DNA repair, enhance ionizing radiation sensitivity, and induce genomic instability in tumorigenesis (7, 17, 35, 70, 76). We have previously reported that specific mutations of S. pombe pol, pol, primase (spp1 and spp2), and ligase (cdc17-K42) confer a mutator phenotype characterized by deletion of sequences flanked by short direct repeats and small sequence alterations (41). Deletion of the fission yeast rad2+ gene, similar to deletion of the homologous gene in budding yeast, RAD27 (71), induces duplication of sequences flanked by short direct repeats (41). Comparison of the panel of replication mutant alleles that confer a mutator phenotype to those that exhibit telomere length alterations shows that all of the replication mutants (especially those implicated in lagging-strand synthesis) that confer a mutator phenotype also exhibit telomere length deregulation (41) (Fig. 1 and 2). However, some replication mutant alleles, such as those in the two pol mutants (polts2 and cdc6-23), and a pol mutant (cdc20-M10), exhibit deregulation of telomere length, but do not display a mutator phenotype (41). These results suggest that the replication genes contribute to telomeric complex stability as well as genomic mutation avoidance in an allele-specific manner. The underlying mechanisms in these mutants causing a mutator phenotype may also differ from those causing telomere deregulation. Studies of budding yeast est1 cells have suggested that telomerase contributes to inhibition of chromosome instability (35). Studies of mammalian cells and rodent models without telomerase RNA have shown that telomerase dysfunction in certain genetic contexts can facilitate cancer development by compromising chromosome integrity (6-8, 17, 37, 76). In this study, we have shown that mutations of replication proteins can affect the homeostasis of telomeres. Using Pol as a paradigm, we demonstrate that mutation of Pol has a significant effect on telomere length maintenance and telomerase protein levels. It is not yet known to what extent the observed mutator phenotypes induced by replication mutants are indirectly contributed by a telomere homeostasis abnormality. Nonetheless, the results of this study and our previous studies (41) suggest that mutations of replication genes, especially those whose products are involved in the lagging-strand synthesis, could have a major impact on the overall genomic stability.
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ACKNOWLEDGMENTS |
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This study was supported by grants from National Cancer Institute of the National Institutes of Health to T.S.W and grants from the Swedish Cancer Fund (2163-B00-11XAC) and the Swedish Research Council (K2002-31X-14197-01A) to P.S. M.D. was a predoctoral fellow supported by the Sweden-America Foundation, the Swedish Institute, and Lennander's Foundation.
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REFERENCES |
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