![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, February 2007, p. 1017-1026, Vol. 27, No. 3
0270-7306/07/$08.00+0 doi:10.1128/MCB.01866-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Jana Paderova,2,
Andrew Wakeham,1,2
Caterina Iorio,1,2
Virginia Zakian,3
Jeremy Squire,2,4 and
Lea Harrington1,2,4*
Campbell Family Institute for Breast Cancer Research, 620 University Avenue, Toronto, Ontario, Canada,1 Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada,2 Princeton University, Lewis Thomas Laboratory, Princeton, New Jersey,3 Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada4
Received 2 October 2006/ Returned for modification 29 October 2006/ Accepted 10 November 2006
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This equilibrium is not simply a push and pull between factors that strictly promote or oppose telomere extension. In S. cerevisiae, the trimeric protein complex composed of Cdc13, Stn1, and Ten1 plays a critical role in the protection of chromosome ends from nucleolytic degradation, and the complex serves both positive and negative roles in the access of telomerase to the telomere during late S phase (24, 27, 66). The Ku70/Ku80 heterodimer, while essential for nonhomologous end joining, also plays a key role both in the recruitment of telomerase to the telomere and in the protection of ends from degradation (9, 25, 65, 74). Several DNA helicases and nucleases are also known to play key roles in the replication and maintenance of telomeres, including Pif1, Sgs1, Mre11, Rtel, and others (20, 34, 41).
Pif1 is a member of a 5'-to-3' DNA helicase family whose other closest members include Rrm3 and, in Schizosaccharomyces pombe, Pfh1 (33, 37, 77). Unlike Rrm3 and Pif1, Pfh1 is essential (77). Rrm3 promotes the progression of replication forks at particular chromosomal loci prone to pausing during DNA replication (including the telomere), and rrm3
cells exhibit a modest telomere lengthening (11, 32, 33, 45, 59, 69). Mutation of ScPif1 leads to telomerase-dependent telomere lengthening (33, 76), and this phenotype depends on the ATP-unwinding activity of ScPif1; the enzyme has a preference for short (<30 nucleotides) RNA-DNA hybrids and can unwind telomerase RNA from a telomeric DNA substrate (15). Thus, Pif1 in S. cerevisiae inhibits telomerase elongation in vivo and in vitro. In humans, overexpression of active PIF1 leads to telomere shortening, and hPIF1 appears to reduce the elongation activity of telomerase in vitro, in a manner consistent with an effect on enzyme processivity (75).
Other roles for Pif1 may reflect a broader role for DNA-unwinding activity at the telomere and in nuclear and mitochondrial DNA replication. In the presence of double-stranded DNA breaks, chromosome ends are rarely healed by the de novo addition of telomeres (54). In the absence of nuclear ScPif1, gross chromosomal rearrangements are increased, and healing of broken ends via telomere addition is more frequent (42, 47, 48, 61). Nuclear ScPif1 is also essential for mitigating the deregulated activity of the RecQ helicase Sgs1 observed in top3 cells (71). Interestingly, ScPif1 (but not Rrm3) colocalizes almost exclusively with Rad52 in nuclei and is recruited to discrete foci after DNA damage (71). ScPif1 and Rrm3 also play roles in the mitochondria, where they contribute to mitochondrial genome stability (21, 37, 51, 52, 67, 70). Finally, both ScPif1 and Pfh1 interact genetically with the Dna2 helicase and cooperate to ensure the correct processing of Okazaki fragments during DNA replication (17, 58).
We took a genetic approach to ascertain the role of Pif1 in mammals and disrupted the one putative murine homologue of ScPif1 and ScRrm3, called mPif1. We found that murine Pif1 expression was highly restricted and was detectable only in highly proliferating cells, such as embryonic stem (ES) cells and murine embryonic fibroblasts (MEFs), and in the hematopoietic lineage. In vitro, murine Pif1 interacted with telomerase activity in a telomerase RNA-independent manner. In mPif1-deficient animals, we found no evidence of sensitivity to DNA damage and no genetic instability, telomere length alteration, or other phenotypic abnormalities in cell types that would normally express Pif1. Moreover, Pif1-deficient animals exhibited normal life spans. These data suggest that that murine Pif1 is dispensable for normal telomere function, development, and aging in inbred mice.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Construction of a murine Pif1 targeting vector.
In brief, PCR primers corresponding to the mPif1 cDNA sequence (GenBank accession no. AY498715) (5'-GGG TAC CAG AAG GTG TTA GAT CTC CTG GAA CAC AAG GTT G-3' [KpnI sense] and 5'-ACT CGA GCG GCC TGC TCT TCA GAA AGC TTC GGT TTG GTG G-3' [XhoI antisense]) were used to amplify an 
3.4-kb long-arm fragment from 200 ng of 129J genomic DNA, using the Expand long-template PCR system (Roche GmbH, Germany). The Expand high-fidelity PCR system (Roche) was used to amplify a 771-bp short arm from 129J Genomic DNA, using the PCR primers 5'-ATC TAG ACC GTG TAT CAA CAC CAG CAT CCT ATT CCA GGT CTG C-3' (XbaI sense) and 5'-CGC GGC CGC ACT GTC TAT AGC CTC AAA GCT GTG TAC ATC ACC TGG-3' (NotI antisense). The resulting PCR products were gel purified using the QiaQuick gel purification system (QIAGEN Inc., Chatsworth, CA), TA cloned into pCR2.1 (Invitrogen, San Diego, CA), and subcloned into pBluescript II KS (Stratagene, La Jolla, CA) containing a phosphoglycerate kinase (PGK)-neomycin cassette. The insert sequence was obtained using fluorescent dideoxynucleotide sequencing and automated detection (ABI/Perkin Elmer, Forest City, CA).
Targeted disruption of the mPif1 gene in ES cells. The mPif1 targeting vector (25 µg) was linearized with the NotI restriction endonuclease at the short arm and electroporated into E14 ES cells (derived from the R129J strain). After G418 selection (300 µg/ml), homologous recombinants were identified by PCR and confirmed by Southern blot analysis, following published protocols (28). Primer mPIF1 Wt-1, which is specific for the region deleted in targeted mPif1, was used to detect the wild-type allele. Primer PGK-1, which is specific for the PGK promoter of the targeting construct, was used to detect the mutant allele. Primer mPIF1 Wt-2 was used to detect both the wild-type and mutant alleles of mPif1. The sequences are as follows: for mPIF1 Wt-1, 5'-GAG GCT GAT CTG ATG ACA GGA TAG CGA G-3'; for PGK-1, 5'-GCT GTC CAT CTG CAC GAG ACT AGT GAG ACG-3'; and for mPIF1 Wt-2, 5'-CAG GGC CTG GGA GTC AGC TCA CCA C-3'. A 912-bp probe flanking the 3' end of the short arm of the targeting vector was used to confirm correct insertion by Southern analysis; this probe was generated by PCR amplification using the following primers: sense, 5' TCT TCA TCG AGA GCC CTG ACA AGC AAC 3'; and antisense, 5' CTG AGG AAG GCA TTG AGA AGT CCC ACC 3'.
Generation of mPif1-deficient mice and ES cells. Chimeric mice were produced by microinjection of independent mPif1+/– ES cell clones into E3.5 C57BL/6J blastocysts and transferred to ICR pseudopregnant foster mothers. The generation of founder mPif1–/– lines and breeding of homozygous-null animals were performed as previously described for mTert–/– animals (22, 38). The initial background is a mixture of 129J and C57BL/6J; therefore, to obtain mPif1–/– in a homogeneous background, the mixed-genetic-background mPif1+/– mice were bred for eight generations to C57BL/6 mice and then mated together to generate mPif1–/– mice (termed G1, C57BL/6). Murine Pif1–/– ES cell clones were generated from G418-resistant mPif1+/– ES cell clones by culturing them with an increased G418 concentration (3.5 mg/ml). The ES cell culture was carried out as described previously (28).
Telomere length analysis. The average telomere fluorescence in populations of isolated splenocytes and thymocytes was measured by fluorescence in situ hybridization (FISH) (57) with minor modifications. A telomere-specific fluorescein isothiocyanate-conjugated (CCCTAA)3 peptide nucleic acid probe (0.3 µg/ml) (Perseptive Biosystems) was used. Telomere fluorescence was expressed as numbers of molecules of equivalent soluble fluorochrome. Quantitative fluorescence in situ hybridization (Q-FISH) of activated splenocytes and MEFs was performed as described previously (14, 78). A Cy3-labeled (CCCTAA)3 peptide nucleic acid (Applied Biosystems) was used as a probe. Chromosomes were counterstained with DAPI (4',6'-diamidino-2-phenylindole). Telomere fluorescence signal intensities were quantified using TFL-Telo Q-FISH software (version 2.1.04.1217; BC Cancer Agency; Steven S. S. Poon and Peter M. Lansdorp). Telomere signal intensities were compiled from at least 10 metaphases (1,600 telomeres) for each sample. Metaphase images from littermates composed of all genotypes (the wild type, mPif1+/–, and mPif1–/–) were analyzed at the same time for technical consistency.
SKY. Spectral karyotyping (SKY) was performed on metaphase spreads from MEFs and splenocyte cells according to the manufacturer's instructions (Applied Spectral Imaging, Carlsbad, CA) and as previously published (4, 60). Metaphase cell spreads, selected for good-quality nonoverlapping chromosome spreads, were analyzed in a blinded manner with activated splenocyte samples from two different +/+, +/–, and –/– murine strains and primary MEF samples from one of each representative genotype (including two separately generated –/– lines). Samples were decoded after image analysis, and data acquisition was complete. According to the International System for Human Cytogenetic Nomenclature (ISCN), karyotypes are normal unless a numerical gain is seen in at least two cells, a chromosomal loss seen in at least three cells, or a chromosomal rearrangement seen in at least two cells (44). DAPI analysis was also used to detect any numerical chromosomal or ploidy changes in MEFs and splenocytes. At least 20 metaphase spreads were analyzed in a blinded manner for at least one sample of each genotype.
Murine splenocyte cytogenetic preparation.
Activated primary mouse splenocytes were isolated from C57BL/6 mice and cultured in RPMI 1640 media containing 10% (vol/vol) fetal calf serum (Sigma Aldrich Co.), 1% (wt/vol) L-glutamine, 0.55 mM ß-mercaptoethanol, and 1% (wt/vol) penicillin-streptomycin (Gibco/BRL, Toronto, Canada). The splenocytes were activated with anti-CD3
antibody (BD Biosciences) for 24 h and then transferred to media supplemented with interleukin 2 (BioSource) for an additional 24 h. The following day, the cultures were treated with 0.1 µg/ml Colcemid (Roche) for 5 to 6 h, swollen in 0.075 M KCl hypotonic buffer, fixed in a 3:1 volume of methanol-acetic acid, and dropped onto slides.
Murine MEF cytogenetic preparation. MEFs were cultured in Dulbecco's modified Eagle's medium-H21 medium containing 10% (vol/vol) fetal calf serum (Sigma Aldrich Co.), 1% (wt/vol) L-glutamine, 0.055 mM ß-mercaptoethanol, and 1% (wt/vol) penicillin-streptomycin (Gibco/BRL, Toronto, Canada). Plates of approximately 80% confluent MEFs were processed in the same manner as the splenocytes except that they were treated with 0.05 µg/ml of Colcemid (Roche) for 3 to 4 h.
Cell viability in splenocytes and MEFs. Freshly isolated mouse splenocytes were harvested from three independent wild-type or mPif1–/– mice and stained with annexin V and 7-amino-actinomycin D according to the manufacturer's instructions (BD Biosciences). In brief, splenocytes were resuspended in binding buffer containing annexin V-PE (to detect apoptosis) and 7-amino-actinomycin D (to detect necrosis) and then processed for flow cytometry analysis. For the analysis of cell viability in MEFs, cells from each genotype (+/+, +/–, or –/–) were seeded at 5 x 104 cells per well, in a total of six wells. The approximate doubling times were as follows: for 2919A, 37 h; for 2945B, 36 h; and for 2945D, 35 h. The R2 values for the exponential curve fits are as follows: for 2919A, 0.9464; for 2945B, 0.9753; and for 2945D, 0.97. The experiment was carried out twice, and no statistical difference in growth rates was observed. For the analysis whose results are shown in Fig. S3B in the supplemental material, exponentially growing MEFs were trypsinized, and the cells were washed and fixed in ice-cold ethanol. For propidium iodide staining, the cells were precipitated and washed with phosphate-buffered saline containing 0.5% (vol/vol) fetal calf serum. The cell pellets were then resuspended in phosphate-buffered saline containing 0.5% (vol/vol) fetal calf serum, 8 µg/ml propidium iodide, and 125 µg/ml RNase A and incubated for 2 h at room temperature in the dark.
|
-Irradiation was administered via a 137Cs source, and UV was administered via a Stratalinker (Stratagene). The cells were then cultured for 3 days, at which time the number of viable cells was determined, in triplicate, by trypan blue exclusion. For the hydroxyurea dose response curve, 1 x 105 passage 5 cells were plated per well in a six-well cluster. Cells were incubated at the indicated concentrations of hydroxyurea for 24 h. The cells were then washed to remove the drug and incubated for 2 days, at which time the number of viable cells was determined, in triplicate, by trypan blue exclusion. Generation of anti-mPif1 antibody. A cDNA fragment encoding the N-terminal 216 amino acids (aa) of mPif1 (aa 1 to 216) was subcloned in-frame with both an amino- and a carboxyl-terminal six-histidine tag into the bacterial expression vector pET30b (Novagen, Madison, WI), purified, and injected into New Zealand White rabbits (University of Toronto). Antibody was affinity purified by incubating 0.5 ml of crude rabbit serum with 200 µg of the same purified mPif1 N-terminal antigen immobilized onto a nitrocellulose filter and eluting the specifically bound antibodies with 50 mM glycine-HCl (pH 2.7). The specificity of this polyclonal antibody was confirmed by Western blot analysis using full-length mPif1 in vitro transcribed and translated in rabbit reticulocytes (Promega) or cell extracts from human 293T cells overexpressing recombinant mPif1.
In vitro transcription and translation. In vitro transcription/translation reactions were performed using the TnT T7 coupled-reticulocyte-lysate system (Promega, Madison, WI) according to the manufacturer's instructions. The reconstitution of murine or human telomerase was performed as described previously (6, 7). The reaction mixtures were then immunoprecipitated with 25 µl of a 50% (vol/vol) slurry of either anti-HA (3F10) agarose beads (Roche), anti-Flag beads (Sigma Aldrich Co.), or protein A-Sepharose-CL4B (Sigma Aldrich Co.) coupled with polyclonal antibody, washed, and resolved as described previously (6, 7). Western blots were probed with anti-HA (3F10), anti-mPif1, or anti-TERT antibody and detected by chemiluminescence (Amersham Pharmacia).
Cell transfection, immunofluorescence, and immunoprecipitation. HA-tagged full-length mPif1 was transfected alone into human 293T cells with FuGene6 (Roche). Exponentially dividing 293T cells transiently expressing vector alone or HA-mPif1 were grown on two-well chamber slides coated with 0.1 mg/ml poly-L-lysine (Sigma, St. Louis, MO), fixed for 10 min in 4% (wt/vol) paraformaldehyde, and stained with DAPI or with affinity purified polyclonal anti-mPif1 primary antibody and an Alexafluor-488 anti-rabbit immunoglobulin G secondary antibody (Molecular Probes). For detection by immunoprecipitation, whole-cell lysis was carried out using 1:4 cell pellet volumes of 0.5% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), in a buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.1 M NaCl, 5 mM ß-mercaptoethanol, and 10% (vol/vol) glycerol. Approximately 1 mg of cell or tissue protein extract was subjected to immunoprecipitation with the appropriate antibody in CHAPS buffer. Beads were washed in the same buffer four times before they were examined by Western analysis.
TRAP. The telomere repeat amplification protocol (TRAP; Intergen, Inc.) was carried out according to the manufacturer's instructions, with minor modifications (35). A cell extract or a portion of beads following immunoprecipitation (typically 2 to 4 µl) was assayed for the presence of telomerase activity. A titration of the cell extract was used to demonstrate that the TRAP products were in the near-linear range.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
4.0 kb present in adult thymuses and in all embryonic tissues and cell lines analyzed (Fig. 2B). The 4-kb mRNA corresponds well with the predicted 3.68-kb full-length mPif1 cDNA (GenBank accession no. AY498715), taking into account a typical poly(A) tail of 200 residues for mature vertebrate mRNAs (72). Disruption of murine Pif1. A targeting construct was designed to delete a region of mPif1 that spanned the first five conserved helicase motifs of mPif1 (Fig. 2A), starting near the end of exon 3 (corresponding to aa 222) and deleting the next five exons (until aa 407), including deletion of a lysine at aa 243 of mPif1, which is predicted to be essential for activity (36). Exons 1, 2, and 8 to 13 remained intact. Note that the neomycin phosphotransferase gene in the disruption cassette is transcribed in a direction opposite that of mPif1, thus decreasing the likelihood of a partial mPif1 transcript after targeted deletion. The linearized mPif1 vector was introduced into E14 (wild-type) ES cells, and after selection in G418, homologous recombinants were identified by PCR and confirmed by Southern blot analysis. Chimeric mice and founder mice were generated as described in Materials and Methods. Germ line transmission of the mutant allele was confirmed by PCR and Southern blot analysis (Fig. 2A). Murine Pif1–/– ES cell clones were generated from G418-resistant mPif1+/– ES cell clones by incubation at an increased G418 concentration (Fig. 2A). Northern analysis of mPif1–/– embryonic stem cells, MEFs, and thymocytes confirmed the complete absence of the mPif1 transcript (Fig. 2B, right).
To determine the tissue distribution of mPif1 protein and confirm its absence in mPif1–/– cells, we generated an antibody to mPif1 (see Fig. S1 in the supplemental material). This antibody was specific to mPif1 expressed in E. coli, rabbit reticulocyte lysates, and 293 cells (see Fig. S1A to C in the supplemental material). Overexpressed mPif1 was detected in 293 cell nuclei; however, the antibody was insufficiently sensitive to detect endogenous mPif1 by immunofluorescence (see Fig. S1C in the supplemental material; also data not shown). A large-scale immunoprecipitation followed by western analysis, using the mPif1 antibody, revealed a faint species at 64 kDa that was present in wild-type and mPif1+/– ES cells but absent in mPif1–/– cells (see Fig. S1B in the supplemental material). Both recombinant mPif1 and the putative endogenous mPif1 species migrated slightly faster (65 kDa) than predicted by its molecular mass of 71 kDa.
Telomere length analysis of mPif1–/– mice. To analyze the consequences of mPif1 deletion upon the length distribution of mammalian telomeres, we mated mPif1–/– animals (as cousins, to prevent spurious effects from unlinked loci) for multiple generations both in the mixed genetic background from which the original knockout strain was derived and after backcrossing mPif1+/– animals into a pure C57BL/6 background. Telomere length was assessed by flow cytometry of fixed cellular DNA hybridized to a fluorescent telomeric probe (flow-FISH) (3) and quantitative measurement of fluorescent telomeric signal at each chromosome end in metaphase preparations (Q-FISH) (Fig. 3 and data not shown) (3). No change in telomere length was observed in mPif1–/– ES cells for up to 100 population doublings (Fig. 3A). In the original mixed genetic background, a statistically significant increase in telomere length was observed in one mPif1–/– litter after four generations; however, this increase was not reproduced in other litters and was no longer evident after five and six mPif1–/– generations (data not shown). After mPif1–/– mice were backcrossed into a pure C57BL/6 background, telomere lengths were again analyzed in successive mPif1–/– generations. No difference in average telomere DNA content or telomere length distribution was observed in mPif1–/– thymocytes or splenocytes for up to four generations (Fig. 3B). Similar results were also obtained after five and six generations in the mPif1–/– background (data not shown). Thus, in stark contrast to the telomere-lengthening phenotype of cells lacking nuclear Pif1 in S. cerevisiae, no change in telomere length was observed for up to six generations of mPif1–/– mice.
|
|
200) (5, 6). Because 100% of the telomerase in the anti-HA immunoprecipitation was associated with HA-Pif1, these results allow us to conclude that neither wild-type nor mutant mPif1 affects telomerase activity when specifically bound to TERT (Fig. 4B). Both full-length hTERT and the 200 hTERT mutant interacted with mPif1 in a telomerase-RNA-independent manner (Fig. 4C, lanes 3 and 4). Testing various truncations of hTERT for binding to mPif1 further emphasized the telomerase RNA independence of this interaction, since the interaction of mPif1 occurred even with hTERT mutants that cannot bind telomerase RNA (e.g., aa 401 to 1132, 536 to 1132, and 401 to 928) (Fig. 4D) (5, 6). Other truncations of hTERT failed to show a specific interaction with mPif1 (data not shown). These data define a putative novel mPif1 interaction domain for hTERT aa 536 to 928. The Pif1-telomerase interaction was specific to HA-mPif1 and not an irrelevant HA-tagged protein of a similar mass (mAif) (Fig. 4D), and the interaction was not sensitive to pretreatment with DNase I (data not shown). Although overexpressed Pif1 could also immunoprecipitate telomerase activity in human cell extracts, the levels of endogenous mPif1 were insufficient to confirm an interaction with endogenous telomerase. However, the absence of mPif1 in cell extracts did not affect telomerase activity in vitro, since extract titrations of mPif1–/– fibroblasts or ES cells, followed by measurement of telomerase activity by the TRAP or the more quantitative, non-PCR elongation assay, revealed no differences in telomerase elongation activity (data not shown).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mammalian Pif1 is associated with telomerase activity when overproduced in rabbit reticulocyte lysates or in cells, and this interaction does not appear to require the telomerase RNA (this study; 43a). In S. cerevisiae, a recent report showed that ScPif1 precipitates the telomerase RNA TLC1, although it was not established whether this fraction of TLC1 was also associated with telomerase activity (23). In that study, four separate point mutations in the region corresponding to the finger domain of Est2 (called est2-up) led to a telomere-lengthening phenotype that depended on nuclear Pif1 (23). We note that the human and murine TERT proteins do not contain significant sequence similarity in the region of the est2-up mutations; thus, it is difficult to make predictions regarding a conserved genetic interaction in this region of TERT. Interestingly, though, this study defines a putative minimal domain of hTERT that interacts with mPif1 and which overlaps with the finger domain. In contrast to results for previous in vitro studies in which purified, recombinant hPIF1 or ScPif1 was added in excess of hTERT or ScEst2 in solution (15, 75), no effect of mPif1 on telomerase activity was seen in this study, even for coimmunoprecipitations where 100% of hTERT/mTert was mPif1 associated. As our analysis of the enzymatic activity of mPif1 is still ongoing, at present we can only infer that mPif1, like its closely related ortholog hPIF1, is active under rabbit reticulocyte lysate conditions permissive for helicase activity. Regardless of the catalytic status of mPif1, we show for the first time that mPif1 can bind to mammalian telomerase in vitro and that this interaction does not depend on the catalytic competence of mPif1. Therefore, it appears that a telomerase-Pif1 interaction is conserved between mammals and S. cerevisiae.
It is possible that the distinct biochemical properties of murine telomerase may mask any effect of Pif1 unwinding activity. Murine telomerase is much less processive than human telomerase (56), and unlike S. cerevisiae, telomerase can dissociate from its substrate (19, 55). The inherent capacity of mTert to release a telomeric substrate could thereby render the unwinding activity of Pif1 redundant. The association of Pif1 with telomerase may become important only in specific circumstances, such as the recruitment of telomerase to broken chromosome ends. Recently, human PIF1 was demonstrated to exhibit an ATP-dependent unwinding activity (29); however, the precise substrate specificities of human and murine Pif1, compared to that of ScPif1, remain to be established.
Consistent with the lack of effect of mPif1 on telomerase activity in vitro or in mPif1–/– extracts, we observed no reproducible change in telomere length in the absence of mPif1 in vivo. Blasco and colleagues also analyzed an independently derived mPif1–/– murine strain and found no observable difference in average telomere lengths or cell viability in response to DNA damage (Maria Blasco, personal communication). In S. cerevisiae, the absence of nuclear Pif1 leads to telomere lengthening (76). In mPif1-deficient mice that already possess long telomeres, it is possible that further telomere lengthening could be offset by the intrastrand excision of telomeric DNA—a phenomenon observed in S. cerevisiae strains with very long telomeres (12, 40) and in human cells deficient in telomere capping (73). It will be interesting to determine whether telomere lengthening in the absence of mPif1 occurs in other genetic backgrounds, particularly in mTert heterozygous animals or outbred murine strains, which possess a shorter average telomere length but retain telomerase activity (22, 38).
The lack of an obvious phenotype in the absence of mPif1 makes it difficult to conclude whether mPif1 is orthologous to S. cerevisiae Pif1 or Rrm3. Homology searches of the murine database did not reveal any other genes with significant similarity to Pif1 or Rrm3. In addition, a search for putative homologs of the known Rrm3-interacting protein Def1, which plays a role in telomere length maintenance (18), did not reveal any proteins with obvious sequence similarity. It is possible that conservation of Pif1 function does not extend to telomere DNA replication in all organisms; however, this conclusion would appear inconsistent with the observation that Pif1 interacts with telomerase (our study; 43a) and with a recent report that human Pif1 is telomere associated (75). Other characterized mammalian helicases do not exhibit extensive redundancy in vivo. For example, the five known RecQ helicases (Wrn, Blm, RecQ4, RecQL, and RecQ5b) and XPB/ERCC3 (implicated in xeroderma pigmentosum and Cockayne syndrome) show limited redundancy in rescuing p53-dependent apoptosis in Bloom-deficient cells (63); however, mutations in each helicase gene have distinct phenotypes in genome stability and DNA replication and repair in vivo and all are separately implicated in human disease (8, 34, 49, 62).
It is quite plausible that mammalian Pif1 function is important for other types of DNA damage not yet examined. For example, loss-of-function studies of the BRCA1-interacting DNA helicase, BACH1, revealed a role in DNA repair distinct from that of BRCA1, and despite sensitivity to cisplatin and mitomycin C, BACH1-deficient cells are relatively unaffected by treatment with UV, X rays, or methyl methanesulfonate (16, 53). To test the role of mPif1 in chromosome healing, we plan to make use of an elegant system developed by Murnane and colleagues, in which a telomere can be cleaved via a unique I-SceI site integrated into the subtelomeric DNA of one chromosome (39, 64). Since deletion of ScPIF1 leads to an increase in mitochondrial DNA point mutations, particularly after oxidative damage (21, 51, 52, 67), it is also possible mPif1 plays a nonessential role in mitochondrial genome stability. Although Scrrm3 cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31, 32, 45, 59, 68, 69). ScRRM3 and ScPIF1 exhibit genetic interactions (including synthetic lethality) with many gene products required in DNA replication and S-phase checkpoint arrest (1, 2, 17, 50, 58, 59, 69, 71). Thus, additional inactivation of other DNA helicases involved in DNA replication or repair or proteins essential for the S-phase checkpoint may yet reveal an interesting phenotype in mPif–/– animals.
|
ACKNOWLEDGMENTS |
|---|
We thank Tak W. Mak for funding Andrew Wakeham's contribution. This study was funded by a grant from the NIH/NIA Longevity Assurance Genes Program (AG024398) and National Cancer Institute of Canada Research grant no. 15072 (using funds from the Canadian Cancer Society) to L.H.
|
FOOTNOTES |
|---|
Published ahead of print on 27 November 2006.
Supplemental material for this article may be found at http://mcb.asm.org/.
These authors contributed equally.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Azam, M., J. Y. Lee, V. Abraham, R. Chanoux, K. A. Schoenly, and F. B. Johnson. 2006. Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence. Nucleic Acids Res. 34:506-516.
3. Baerlocher, G. M., J. Mak, T. Tien, and P. M. Lansdorp. 2002. Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls. Cytometry 47:89-99.[CrossRef][Medline]
4. Bayani, J., M. Zielenska, P. Marrano, Y. Kwan Ng, M. D. Taylor, V. Jay, J. T. Rutka, and J. A. Squire. 2000. Molecular cytogenetic analysis of medulloblastomas and supratentorial primitive neuroectodermal tumors by using conventional banding, comparative genomic hybridization, and spectral karyotyping. J. Neurosurg. 93:437-448.[Medline]
5. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 2001. Functional multimerization of the human telomerase reverse transcriptase. Mol. Cell. Biol. 21:6151-6160.
6. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 2000. Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding. Mol. Biol. Cell 11:3329-3340.
7. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 1998. Reconstitution of human telomerase activity in vitro. Curr. Biol. 8:177-180.[CrossRef][Medline]
8. Bennett, R. J., and J. L. Keck. 2004. Structure and function of RecQ DNA helicases. Crit. Rev. Biochem. Mol. Biol. 39:79-97.[CrossRef][Medline]
9. Bertuch, A. A., and V. Lundblad. 2003. Which end: dissecting Ku's function at telomeres and double-strand breaks. Genes Dev. 17:2347-2350.
10. Bessler, J. B., J. Z. Torredagger, and V. A. Zakian. 2001. The Pif1p subfamily of helicases: region-specific DNA helicases? Trends Cell Biol. 11:60-65.[CrossRef][Medline]
11. Bessler, J. B., and V. A. Zakian. 2004. The amino terminus of the Saccharomyces cerevisiae DNA helicase Rrm3p modulates protein function altering replication and checkpoint activity. Genetics 168:1205-1218.
12. Bhattacharyya, M. K., and A. J. Lustig. 2006. Telomere dynamics in genome stability. Trends Biochem. Sci. 31:114-122.[CrossRef][Medline]
13. Biek, D. P., and S. N. Cohen. 1986. Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. J. Bacteriol. 167:594-603.
14. Blasco, M. A., H. W. Lee, M. P. Hande, E. Samper, P. M. Lansdorp, R. A. DePinho, and C. W. Greider. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91:25-34.[CrossRef][Medline]
15. Boule, J. B., L. R. Vega, and V. A. Zakian. 2005. The yeast Pif1p helicase removes telomerase from telomeric DNA. Nature 438:57-61.[CrossRef][Medline]
16. Bridge, W. L., C. J. Vandenberg, R. J. Franklin, and K. Hiom. 2005. The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair. Nat. Genet. 37:953-957.[CrossRef][Medline]
17. Budd, M. E., C. C. Reis, S. Smith, K. Myung, and J. L. Campbell. 2006. Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol. Cell. Biol. 26:2490-2500.
18. Chen, Y. B., C. P. Yang, R. X. Li, R. Zeng, and J. Q. Zhou. 2005. Def1p is involved in telomere maintenance in budding yeast. J. Biol. Chem. 280:24784-24791.
19. Cohn, M., and E. H. Blackburn. 1995. Telomerase in yeast. Science 269:396-400.
20. Ding, H., M. Schertzer, X. Wu, M. Gertsenstein, S. Selig, M. Kammori, R. Pourvali, S. Poon, I. Vulto, E. Chavez, P. P. Tam, A. Nagy, and P. M. Lansdorp. 2004. Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell 117:873-886.[CrossRef][Medline]
21. Doudican, N. A., B. Song, G. S. Shadel, and P. W. Doetsch. 2005. Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae. Mol. Cell. Biol. 25:5196-5204.
22. Erdmann, N., Y. Liu, and L. Harrington. 2004. Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc. Natl. Acad. Sci. USA 101:6080-6085.
23. Eugster, A., C. Lanzuolo, M. Bonneton, P. Luciano, A. Pollice, J. F. Pulitzer, E. Stegberg, A. S. Berthiau, K. Forstemann, Y. Corda, J. Lingner, V. Geli, and E. Gilson. 2006. The finger subdomain of yeast telomerase cooperates with Pif1p to limit telomere elongation. Nat. Struct. Mol. Biol. 13:734-739.[CrossRef][Medline]
24. Evans, S. K., and V. Lundblad. 2000. Positive and negative regulation of telomerase access to the telomere. J. Cell Sci. 113:3357-3364.[Abstract]
25. Fisher, T. S., and V. A. Zakian. 2005. Ku: a multifunctional protein involved in telomere maintenance. DNA Repair (Amsterdam) 4:1215-1226.[CrossRef]
26. Gorbalenya, A. E., and E. V. Koonin. 1993. Helicases: amino acid sequence comparisons and structure-function relationships. Curr. Opin. Struct. Biol. 3:419-429.
27. Grandin, N., C. Damon, and M. Charbonneau. 2001. Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J. 20:1173-1183.[CrossRef][Medline]
28. Hakem, R., J. L. de la Pompa, C. Sirard, R. Mo, M. Woo, A. Hakem, A. Wakeham, J. Potter, A. Reitmair, F. Billia, E. Firpo, C. C. Hui, J. Roberts, J. Rossant, and T. W. Mak. 1996. The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell 85:1009-1023.[CrossRef][Medline]
29. Huang, Y., D. H. Zhang, and J. Q. Zhou. 2006. Characterization of ATPase activity of recombinant human Pif1. Acta Biochim. Biophys. Sin. (Shanghai) 38:335-341.
30. Hug, N., and J. Lingner. 2006. Telomere length homeostasis. Chromosoma 115:413-425.
31. Ivessa, A. S., B. A. Lenzmeier, J. B. Bessler, L. K. Goudsouzian, S. L. Schnakenberg, and V. A. Zakian. 2003. The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes. Mol. Cell 12:1525-1536.[CrossRef][Medline]
32. Ivessa, A. S., J. Q. Zhou, V. P. Schulz, E. K. Monson, and V. A. Zakian. 2002. Saccharomyces Rrm3p, a 5' to 3' DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA. Genes Dev. 16:1383-1396.
33. Ivessa, A. S., J. Q. Zhou, and V. A. Zakian. 2000. The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA. Cell 100:479-489.[CrossRef][Medline]
34. Khakhar, R. R., J. A. Cobb, L. Bjergbaek, I. D. Hickson, and S. M. Gasser. 2003. RecQ helicases: multiple roles in genome maintenance. Trends Cell Biol. 13:493-501.[CrossRef][Medline]
35. Kim, N. W., M. A. Piatyszek, K. R. Prowse, C. B. Harley, M. D. West, P. L. Ho, G. M. Coviello, W. E. Wright, S. L. Weinrich, and J. W. Shay. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011-2015.
36. Korolev, S., J. Hsieh, G. H. Gauss, T. M. Lohman, and G. Waksman. 1997. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90:635-647.[CrossRef][Medline]
37. Lahaye, A., H. Stahl, D. Thines-Sempoux, and F. Foury. 1991. PIF1: a DNA helicase in yeast mitochondria. EMBO J. 10:997-1007.[Medline]
38. Liu, Y., H. Kha, M. Ungrin, M. O. Robinson, and L. Harrington. 2002. Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert. Proc. Natl. Acad. Sci. USA 99:3597-3602.
39. Lo, A. W., C. N. Sprung, B. Fouladi, M. Pedram, L. Sabatier, M. Ricoul, G. E. Reynolds, and J. P. Murnane. 2002. Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells. Mol. Cell. Biol. 22:4836-4850.
40. Lustig, A. J. 2003. Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion. Nat. Rev. Genet. 4:916-923.[CrossRef][Medline]
41. Lydall, D. 2003. Hiding at the ends of yeast chromosomes: telomeres, nucleases and checkpoint pathways. J. Cell Sci. 116:4057-4065.
42. Mangahas, J. L., M. K. Alexander, L. L. Sandell, and V. A. Zakian. 2001. Repair of chromosome ends after telomere loss in Saccharomyces. Mol. Biol. Cell 12:4078-4089.
43. Marchler-Bauer, A., J. B. Anderson, P. F. Cherukuri, C. DeWeese-Scott, L. Y. Geer, M. Gwadz, S. He, D. I. Hurwitz, J. D. Jackson, Z. Ke, C. J. Lanczycki, C. A. Liebert, C. Liu, F. Lu, G. H. Marchler, M. Mullokandov, B. A. Shoemaker, V. Simonyan, J. S. Song, P. A. Thiessen, R. A. Yamashita, J. J. Yin, D. Zhang, and S. H. Bryant. 2005. CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res. 33:D192-D196.
43. Mateyak, M. K., and V. A. Zakian. 2006. Human PIF1 helicase is cell cycle regulated and associates with telomerase. Cell Cycle 5:2796-2804.[Medline]
44. Mitelman, F. 1995. An international system for human cytogenetic nomenclature. S. Karger, Basel, Switzerland.
45. Mohanty, B. K., N. K. Bairwa, and D. Bastia. 2006. The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103:897-902.
46. Myers, R. S., and F. W. Stahl. 1994. Chi and the RecBC D enzyme of Escherichia coli. Annu. Rev. Genet. 28:49-70.[Medline]
47. Myung, K., C. Chen, and R. D. Kolodner. 2001. Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Nature 411:1073-1076.[CrossRef][Medline]
48. Myung, K., V. Pennaneach, E. S. Kats, and R. D. Kolodner. 2003. Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability. Proc. Natl. Acad. Sci. USA 100:6640-6645.
49. Oh, K. S., S. G. Khan, N. G. Jaspers, A. Raams, T. Ueda, A. Lehmann, P. S. Friedmann, S. Emmert, A. Gratchev, K. Lachlan, A. Lucassan, C. C. Baker, and K. H. Kraemer. 2006. Phenotypic heterogeneity in the XPB DNA helicase gene (ERCC3): xeroderma pigmentosum without and with Cockayne syndrome. Hum. Mutat. 27:1092-1103[CrossRef][Medline]
50. Ooi, S. L., D. D. Shoemaker, and J. D. Boeke. 2003. DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat. Genet. 35:277-286.[CrossRef][Medline]
51. O'Rourke, T. W., N. A. Doudican, M. D. Mackereth, P. W. Doetsch, and G. S. Shadel. 2002. Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins. Mol. Cell. Biol. 22:4086-4093.
52. O'Rourke, T. W., N. A. Doudican, H. Zhang, J. S. Eaton, P. W. Doetsch, and G. S. Shadel. 2005. Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability. Gene 354:86-92.[CrossRef][Medline]
53. Peng, M., R. Litman, Z. Jin, G. Fong, and S. B. Cantor. 2006. BACH1 is a DNA repair protein supporting BRCA1 damage response. Oncogene 25:2245-2253.[CrossRef][Medline]
54. Pennaneach, V., C. D. Putnam, and R. D. Kolodner. 2006. Chromosome healing by de novo telomere addition in Saccharomyces cerevisiae. Mol. Microbiol. 59:1357-1368.[CrossRef][Medline]
55. Prescott, J., and E. H. Blackburn. 1997. Functionally interacting telomerase RNAs in the yeast telomerase complex. Genes Dev. 11:2790-2800.
56. Prowse, K. R., A. A. Avilion, and C. W. Greider. 1993. Identification of a nonprocessive telomerase activity from mouse cells. Proc. Natl. Acad. Sci. USA 90:1493-1497.
57. Rufer, N., W. Dragowska, G. Thornbury, E. Roosnek, and P. M. Lansdorp. 1998. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat. Biotechnol. 16:743-747.[CrossRef][Medline]
58. Ryu, G. H., H. Tanaka, D. H. Kim, J. H. Kim, S. H. Bae, Y. N. Kwon, J. S. Rhee, S. A. MacNeill, and Y. S. Seo. 2004. Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast. Nucleic Acids Res. 32:4205-4216.
59. Schmidt, K. H., and R. D. Kolodner. 2004. Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase. Mol. Cell. Biol. 24:3213-3226.
60. Schrock, E., T. Veldman, H. Padilla-Nash, Y. Ning, J. Spurbeck, S. Jalal, L. G. Shaffer, P. Papenhausen, C. Kozma, M. C. Phelan, E. Kjeldsen, S. A. Schonberg, P. O'Brien, L. Biesecker, S. du Manoir, and T. Ried. 1997. Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum. Genet. 101:255-262.[CrossRef][Medline]
61. Schulz, V. P., and V. A. Zakian. 1994. The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation. Cell 76:145-155.[CrossRef][Medline]
62. Sharma, S., K. M. Doherty, and R. M. Brosh, Jr. 2006. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability. Biochem. J. 398:319-337.[CrossRef][Medline]
63. Spillare, E. A., X. W. Wang, C. von Kobbe, V. A. Bohr, I. D. Hickson, and C. C. Harris. 2006. Redundancy of DNA helicases in p53-mediated apoptosis. Oncogene 25:2119-2123.[CrossRef][Medline]
64. Sprung, C. N., G. Afshar, E. A. Chavez, P. Lansdorp, L. Sabatier, and J. P. Murnane. 1999. Telomere instability in a human cancer cell line. Mutat. Res. 429:209-223.[Medline]
65. Stellwagen, A. E., Z. W. Haimberger, J. R. Veatch, and D. E. Gottschling. 2003. Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev. 17:2384-2395.
66. Taggart, A. K., and V. A. Zakian. 2003. Telomerase: what are the Est proteins doing? Curr. Opin. Cell Biol. 15:275-280.[CrossRef][Medline]
67. Taylor, S. D., H. Zhang, J. S. Eaton, M. S. Rodeheffer, M. A. Lebedeva, W. O'Rourke, T. W. Siede, and G. S. Shadel. 2005. The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae. Mol. Biol. Cell 16:3010-3018.
68. Torres, J. Z., J. B. Bessler, and V. A. Zakian. 2004. Local chromatin structure at the ribosomal DNA causes replication fork pausing and genome instability in the absence of the S. cerevisiae DNA helicase Rrm3p. Genes Dev. 18:498-503.
69. Torres, J. Z., S. L. Schnakenberg, and V. A. Zakian. 2004. Saccharomyces cerevisiae Rrm3p DNA helicase promotes genome integrity by preventing replication fork stalling: viability of rrm3 cells requires the intra-S-phase checkpoint and fork restart activities. Mol. Cell. Biol. 24:3198-3212.
70. Van Dyck, E., F. Foury, B. Stillman, and S. J. Brill. 1992. A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. EMBO J. 11:3421-3430.[Medline]
71. Wagner, M., G. Price, and R. Rothstein. 2006. The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae. Genetics 174:555-573
72. Wahle, E., and W. Keller. 1996. The biochemistry of polyadenylation. Trends Biochem. Sci. 21:247-250.[CrossRef][Medline]
73. Wang, R. C., A. Smogorzewska, and T. de Lange. 2004. Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119:355-368.[CrossRef][Medline]
74. Williams, B., and A. J. Lustig. 2003. The paradoxical relationship between NHEJ and telomeric fusion. Mol. Cell 11:1125-1126.[CrossRef][Medline]
75. Zhang, D. H., B. Zhou, Y. Huang, L. X. Xu, and J. Q. Zhou. 2006. The human Pif1 helicase, a potential Escherichia coli RecD homologue, inhibits telomerase activity. Nucleic Acids Res. 34:1393-1404.
76. Zhou, J., E. K. Monson, S. C. Teng, V. P. Schulz, and V. A. Zakian. 2000. Pif1p helicase, a catalytic inhibitor of telomerase in yeast. Science 289:771-774.
77. Zhou, J. Q., H. Qi, V. P. Schulz, M. K. Mateyak, E. K. Monson, and V. A. Zakian. 2002. Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases. Mol. Biol. Cell 13:2180-2191.
78. Zijlmans, J. M., U. M. Martens, S. S. Poon, A. K. Raap, H. J. Tanke, R. K. Ward, and P. M. Lansdorp. 1997. Telomeres in the mouse have large interchromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. USA 94:7423-7428.
| ||||||
-HA) immunoprecipitations (2 µl) of the same lysates as those used for lanes 3 and 4, respectively. Lanes 7 to 10, duplicates of lanes 3 to 6. The expression of HA-tagged mPif1 was confirmed by anti-HA Western blotting (bottom panel). In this experiment, mTert was untagged and undetectable with available antibodies. All TRAP reaction mixtures contained an internal control product to indicate PCR amplification (IC) (arrow at right). (B and C) Murine Pif1 interacts with human telomerase. (B) TRAP analysis of 0.5 µl of the RRLs from Fig.