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[Cancer Research 64, 3830-3837, June 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

DNA Polymerase ß Interacts with TRF2 and Induces Telomere Dysfunction in a Murine Mammary Cell Line

Poppy Fotiadou1, Octavian Henegariu2 and Joann B. Sweasy1

Departments of 1 Therapeutic Radiology and 2 Immunobiology, Yale University School of Medicine, New Haven, Connecticut


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA polymerase ß (Polß) is a DNA repair protein that functions in base excision repair and meiosis. The enzyme has deoxyribose phosphate lyase and polymerase activity, but it is error prone because it bears no proofreading activity. Errors in DNA repair can lead to the accumulation of mutations and consequently to tumorigenesis. Polß expression has been found to be higher in tumors, and deregulation of its expression has been found to induce chromosomal instability, a hallmark of tumorigenesis, but the underlying mechanisms are unclear. In the present study, we have investigated whether ectopic expression of Polß influences the stability of chromosomes in a murine mammary cell line. The results demonstrate a telomere dysfunction phenotype: an increased rate of telomere loss and chromosome fusion, suggesting that ectopic expression of Polß leads to telomere dysfunction. In addition, Polß interacts with TRF2, a telomeric DNA binding protein. Colocalization of the two proteins occurs at nontelomeric sites and appears to be influenced by the change in the status of the telomeric complex.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA repair pathways and telomere maintenance are prominent among the molecular machinery designed to counteract the genetic instability driving tumorigenesis (1) . DNA polymerase ß (Polß) is a key enzyme of the base excision repair (BER) pathway (2) . During BER, following glycosylase and AP endonuclease activity, Polß excises the deoxyribose phosphate (dRP) moiety and catalyzes DNA repair synthesis by filling single nucleotide gaps (short-patch BER) or by displacing the downstream DNA strand (long-patch BER; Refs. 3 , 4 ). Consistent with the critical role of Polß in BER-dependent maintenance of genomic integrity (5) , Polß-knockout mice are embryonic lethal, whereas the fibroblasts derived from the mice are hypersensitive to alkylating agents and oxidative stress (6, 7, 8) . The Polß gene encodes a Mr 39,000 protein with two distinct domains: the amino-terminal dRP lyase domain (Mr 8,000) and the COOH-terminal polymerase domain (Mr 31,000; Ref. 9 ). Polß is ubiquitously distributed and highly conserved among eukaryotes and is expressed in all tissues and invariably throughout the cell cycle (4) . Additional reports provide evidence that Polß plays a role in meiosis (10 , 11) and in mammalian DNA replication (12) . Furthermore, Reichenberger and Pfeiffer (13) have reported that Polß has a role in nonhomologous DNA end joining in Xenopus laevis.

Expression of Polß is altered in tumor cells. High levels of Polß have been detected in ovarian tumors (14) and in prostate, breast, and colon cancer tissues in which the protein was 11-, 286-, and 22-fold higher, respectively, as compared with adjacent normal tissues (15) . A splice variant of Polß also has been found to be in tumors, and point mutations of Polß have been identified in prostate, colon, and gastric carcinomas (16, 17, 18) . It also has been reported that overexpression of the wild-type Polß induces chromosomal instability (19 , 20) .

Telomeres, the natural ends of mammalian chromosomes, have been proposed to be biologic determinants in the process of tumorigenesis (21) . Their primary role is to protect chromosome ends from recombination, fusion, and recognition as damaged DNA (22) . Human and mouse telomeres consist of several kilobases of tandem TTAGGG repeats. This sequence is maintained by telomerase, a reverse transcriptase that adds the repeats onto the 3' protrusions (G-overhangs) of chromosomes (22 , 23) . These DNA repeats also provide a landing platform for a suite of telomere-associated proteins, including telomere-specific proteins TRF1 and TRF2, histones, tankyrases, and DNA repair factors such as Ku, DNA-PKcs, and Mre11-Rad50-NBS1 (24, 25, 26, 27, 28, 29) .

Recent studies suggest that the protection of human chromosome ends primarily depends on the telomeric protein TRF2 (29) . TRF2 is a ubiquitously expressed DNA binding protein that binds directly to the tandem array of duplex TTAGGG repeats of all of the human telomeres at all of the stages of the cell cycle (30 , 31) . Intriguingly, this protein can remodel telomeric DNA into t-loops (32) . T-loops are lariat-like structures that appear to be formed by the invasion of the 3' overhang into the duplex part of the telomeric repeat array (D-loop; Refs. 32 , 33 ). TRF2 is bound at the base of the loop (31) . Inhibition of TRF2 in cultured human cells leads to multilevel telomere dysfunction and at the cellular level induces ATM and p53-mediated apoptosis and senescence (25 , 27 , 34) . At the chromosomal level, it generates end-to-end fusions, and the telomeres lose their 3' G-overhangs (34, 35, 36) . To ensure efficient telomere protection, TRF2 has acquired the help of several known binding partners, including the Mre11 complex and hRap1 (37 , 38) .

Despite the fact that DNA repair processes are normally prohibited from acting on telomeres, a growing body of evidence has led to the initially counterintuitive notion that repair and checkpoint proteins function in normal telomere maintenance (28 , 39, 40, 41) . For example, in mammalian cells, abnormalities in telomere length and function have been observed in DNA-PKcs-deficient cells (26 , 42 , 43) , Ku-deficient cells (44 , 45) , and cells from patients with Werner’s syndrome (46 , 47) , Bloom’s syndrome, Fanconi anemia, and ataxia telangiectasia (48) .

We show that ectopic expression of Polß induces a telomere dysfunction phenotype that is manifested as end-to-end fusions and chromosomal instability. Moreover, Polß physically associates with TRF2 in human and mouse cells. This association is localized to nontelomeric foci, and it appears to be influenced by the telomere status of the cell.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction.
The pIREShyg2 vector used in these studies was obtained from Clontech (Palo Alto, CA). This vector contains an attenuated version of the internal ribosome entry site (IRES) for the encephalomyocarditis virus, which permits the gene of interest and the hygromycin B selection marker to be translated from a single mRNA. Therefore, cells that are resistant to hygromycin B are most likely expressing the gene of interest. Wild-type (WT) Polß was subcloned into the BsrG1 and NheI sites of this vector using standard methods (49) . The hemagglutinin (HA) epitope tag was subcloned along with the WT gene at 3' ends; therefore, on expression, the Polß proteins would be fused to the HA epitope. We have demonstrated previously that the Polß-HA fusion protein is active in a standard polymerase assay. The fusion of HA to Polß permits us to examine specifically expression levels of the enzyme that was introduced into the cells (50) . The DNA sequences of the inserts and cloning junctions were determined for each construct before use.

Cell Lines and Culture Conditions.
C127 cells were obtained from American Type Culture Collection (Manassas, VA). C127 is a nontransformed clonal line derived from the mammary tissue of an RII mouse (51) . The C127 cells were transfected with the pIREShyg2 vector alone or with the WT construct using FuGENE 6 (Roche, Basel, Switzerland), and clones were isolated by limiting dilution in DMEM supplemented with 5% fetal bovine serum, 900 µg/ml G-418, and 300 µg/ml hygromycin-B. Thereafter, cells were maintained in this medium but with 250 µg/ml hygromycin-B. In this way, two clonal cell lines (C127-Polß) expressing full-length Polß and a control C127 cell line (C127-IRES) containing the backbone vector were generated. Hela1.2.11 and HelaS3 were grown in DMEM supplemented with penicillin-streptomycin and 10% fetal bovine serum (Life Technologies, Rockville, MD). HelaLxSn and HelahTERT were grown in DMEM supplemented with penicillin-streptomycin, 10% fetal bovine serum (Life Technologies), 10 mM HEPES, and 500 µg/ml G418.

Whole Cell Extracts.
Cells grown in T75-cm2 flasks were harvested by trypsinization, washed with 10 ml PBS, and were collected after centrifugation at 1000 x g for 5 min. Whole cell protein extracts were made using the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL) according to manufacturer’s instructions. Protein concentrations were determined by using the bicinchonic acid (BCA) protein assay with BSA as a standard.

Immunoprecipitation and Western Blot Analysis.
Protein lysates (1 mg) were incubated with anti-TRF2 (2 µg/ml) or anti-Polß (2 µg/ml) at 4°C with rocking for 1 h. Antigen-antibody complexes were collected on protein A beads, washed three times with TNE buffer [100 mM Tris (pH 7.5), 150 mM NaCl, and 0.2 mM EGTA] and suspended in Laemmli buffer. Following boiling, samples were fractionated, and proteins were transferred to polyvinylidene difluoride by semidry blotting. After blocking in PBS containing 5% nonfat milk powder and 0.5% Tween 20, blots were incubated for 12–16 h at 4°C with the monoclonal antibody (mAb) anti-TRF2 (IMG24; Imgenex, San Diego, CA) or with the mAb anti-Polß antibody (Novus Biologicals, Littleton, CO), followed by horseradish peroxidase-conjugated donkey antimouse IgG (Amersham Biosciences, Piscataway, NJ; 1:10,000). The binding of the secondary antibody was detected using the enhanced chemiluminescence kit (Amersham Biosciences).

To determine whether the hygromycin-B-resistant cells were expressing Polß protein fused to the HA epitope, cell extracts were prepared from each cell line and subjected to Western blot analysis. Approximately 1 x 104 cells were seeded into a T25 flask and grown to confluence. The cells were washed in PBS and harvested by trypsinization. After washing in PBS, the cells were resuspended in buffer A [50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM Na2S2O5, 1 µG/ml pepstatin A, and 0.5 M NaCl], incubated on ice for 5 min, and sonicated on ice three times for 10 s. After boiling in loading buffer, ~500 µG of extract were resolved by electrophoresis, blotted to nitrocellulose, and incubated with a 1:1000 dilution of mAb raised against the HA epitope (Covance Research Products, Inc., Berkeley, CA) as described previously (50) .

Transient Transfections.
For transient transfections into 293T cells, the full-length hTRF2 was inserted into the ClaI and NotI sites of pEBG vector (pEBGhTRf2; New England Biolabs, Beverly, MA), and constructs were sequenced before use. Logarithmically growing 293T cells were transiently transfected by calcium precipitation with pEBGhTRF2 and/or the expression vector pEBG alone. Twenty-four h later, transfectants were treated with DMEM containing 10% fetal bovine serum for another 24 h before being processed for immunoprecipitations.

Yeast Two-Hybrid Assay.
LexA-Polß clones were generated by PCR amplification of DNA sequences encoding the indicated amino acids from a plasmid containing the full-length Polß, followed by insertion into the Xma and SalI sites of vector pGBKT7. GAD-TRF2 clones were generated by PCR amplification of HeLa cDNA and inserted into the BamH1 and EcoRI sites of vector pGADT7. All of the constructs were sequenced before use. The host strain Y130 [MATa his3D200trp1-901 leu 2-3, 112ade2 LYS:: (lexAop)4-HIS3URA3::(lexAop)8-lacZ] was transformed using LiAc (52) . The Polß-interacting protein XRCC1 was used as a positive control. ß-Galactosidase activities were measured essentially as described except that cells were disrupted by freeze-thawing using liquid nitrogen (53) .

Metaphase Spreads.
C127 cells and primary mouse embryonic fibroblasts (MEFs) were incubated with 0.5 µg Colcemid/ml of growth media at 37°C for 2–3 h, harvested by trypsinization, and hypotonically swollen in 0.075 M KCl. Cells subsequently were conditionally fixed in methanol-acetic acid (3:1) and were dropped onto water-wetted slides and Giemsa stained. Images were acquired on a Zeiss Axioplan 2 microscope (Oberkochen, Germany) and processed using CytoVision software (Applied Imaging, Santa Clara, CA).

Indirect Immunofluorescence and Fluorescence in Situ Hybridization.
Cells growing in LabTek four-well slides were washed in PBS, fixed in 100% ethanol (10 min), and permeabilized with 0.5% NP40 in PBS for 10 min. Samples were blocked with fish gelatin in PBS and processed for fluorescence in situ hybridization. The telomeric probe was generated by PCR labeling, mixing two long oligonucleotides, (CCCTAA)7 and (TTAGGG)7, at 1 µM each and running 30 PCR cycles in the presence of 1:2 biotin-dUTP:dTTP and genomic DNA template (54) . Cells were denatured 10–15 min at 65°C in the presence of 50–100 ng biotinylated probe, and hybridization was performed overnight at 37°C. After washing three times in 2x SSC, cells were processed for indirect immunofluorescence using a mouse anti-TRF2 mAb (IMG24; Imgenex) and a rabbit polyclonal IgG for Polß (11) . Primary antibodies were detected with tetramethyl rhodamine isothiocyanate-conjugated antimouse (for TRF2) and FITC-donkey antirabbit (for Polß; The Jackson Laboratory, Bar Harbor, ME). Telomeric TTAGGG repeats were detected with Cy5-conjugated antiavidin, and DNA was stained with 4,6-diamino-2-phenyllndole (0.2 mg/ml). Images of every fluorescent channel were taken on an Olympus Provis microscope (Tokyo, Japan), equipped with a Photometrix SenSys camera (Photometrix, Tucson, AZ) and appropriate filter sets, and using PowerGene image-capturing software (Vysis, Downers Grove, IL).

Telomere Length Assay.
Genomic DNA was isolated as described previously and cleaved with HinfI and RsaI overnight at 37°C (29) . Approximately 5 µg DNA were fractionated on 0.7% agarose and Southern transferred to a nylon membrane, and telomeric restriction fragments were detected with an end-labeled TTAGGG probe as described previously (34) . The median length of the telomeric restriction fragments was determined using ImageQuant software (Amersham Biosciences) after scanning with PhosphoImager (STORM 860; Molecular Dynamics, Sunnyvale, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Polß Overexpression Induces Chromosome End Fusions in Murine C127 Cells.
Polß overexpression is a common finding in a number of tumors. In an attempt to study the consequences of deregulation of Polß in the genomic instability of C127 murine mammary fibroblasts, an expression vector harboring the full-length rat DNA Polß (C127-Polß) cDNA was transfected into C127 cells. Stable hygromycin B-resistant colonies were isolated and expanded into cell lines. The ectopic expression of Polß in C127 cells was verified by Western blot analysis, as shown in Fig. 1Citation . The monoclonal antibody raised against the HA epitope recognizes only the ectopically expressed Polß protein in the C127-Polß cells (Fig. 1Citation , Lanes 2 and 3). None of this protein is detected in C127-IRES cells, which contain the empty vector.



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  		Fig. 1. Polymerase ß (Polß) is ectopically expressed in C127-transfected cells. Cell extracts were prepared as described and immunostained as described in "Materials and Methods." Lane 1 is protein extract from C127 cells containing pIREShygB2 alone. Lanes 2 and 3 are protein extracts isolated from the stable cell lines C127-Polß #1 and C127-Polß #2, respectively. The membrane was incubated with antibody raised against the hemagglutinin (HA) epitope and should recognize proteins expressed from the pIREShygB construct and not endogenous proteins. The molecular weight of Polß-HA proteins was ~Mr 40,000 as expected.

 
The effects of such an expression then were analyzed. Whereas overexpression of the full-length Polß had no significant effect on the short-term growth of C127 cells, by passage 8, ~20% of the cells became enlarged and had a vacuolated cytoplasm, as shown in Fig. 2Citation , resembling senescent cells. However, we were unable to detect expression of SA-ß-galactosidase, a marker of senescence, in these cells (data not shown).



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  		Fig. 2. Ectopic expression of polymerase ß (Polß) protein results in morphologic changes of C127cells. As passage number increased C127, cells transfected with full-length Polß showed giant cells with a vacuolated cytoplasm. A, an example of a lawn of C127-Polß cells that contains giant cells at 10x magnification. B, a giant cell at 20x magnification. C, close-up view of a different giant cell (20x magnification).

 
We also examined the chromosomes of cells expressing ectopic Polß. C127 cells expressing exogenous Polß contained large numbers of telomere fusions, as shown in Fig. 3BCitation , when compared with cells containing the IRES vector alone (Fig. 3A)Citation . Approximately 60% of the IRES cells have between two and nine fusions/cell, suggesting that C127 cells themselves have an inherent defect leading to telomere fusions, as shown in Fig. 3ECitation . However, 32% of cells containing the backbone vector alone (IRES) have no telomere fusions, whereas only 4% of cells expressing WT Polß have no fusions at their telomeres. In addition, 35% of the 120 cells we examined that express WT Polß have >=10 fusions/cell, whereas none of the 90 IRES cells we examined contain >=10 fusions/cell.



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  		Fig. 3. Polymerase ß (Polß) overexpression induces metaphase fusions in C127 mouse cells. A, a representative metaphase cell from C127-internal ribosome entry site (IRES) cells showing normal chromosomes. B, a representative metaphase cell from C127-Polß cells. Note the fused chromosomes as depicted by the arrow. C, a metaphase cell from primary mouse embryonic fibroblasts (MEFs) derived from wild-type mouse. D, a representative metaphase cell from MEFs derived from a Polß knockout mouse. The arrows denote breaks. E, histogram representation of the fusions seen in C127-Polß and C127-IRES metaphases. The black and white bars represent C127-Polß and C127-IRES, respectively.

 
Polß is a DNA repair enzyme, and any deregulation of this enzyme is expected to have a direct effect on the efficiency of the DNA repair machinery. Reduced accuracy of DNA repair could eventually lead to increased levels of DNA damage. Fusion of chromosome ends has been documented in cells containing DNA damage. Therefore, we asked whether the cell responds in the same way to a different form of Polß deregulation, namely, deletion of the Polß gene. To examine this, metaphase spreads of MEFs derived from mPolß–/– and mPolß+/+ littermates were assayed for chromosomal aberrations. In contrast to cells expressing exogenous Polß, MEFs obtained from Polß–/– embryos had extensive chromosomal damage, including a median of 4.74 breaks/metaphase nucleus, as depicted by the arrows in Fig. 3DCitation . However, no telomeric fusions were observed, as shown in Fig. 3, C and DCitation . In aggregate, the cytogenetic analysis indicated that absence and/or deregulation of Polß causes chromosomal instability. Notably, however, ectopic expression of Polß results in end-to-end fusions, whereas deletion of Polß results in chromosome breaks.

Deregulation of Polß Alters Telomere Length.
Chromosome end fusions are a hallmark of telomere dysfunction that are usually manifested as telomere length alterations. To determine whether the end fusions in C127 Polß-overexpressing cells altered telomere length, we sought physical evidence for telomere fusion in naked genomic DNA. Detection of telomeric restriction fragments in genomic DNA from control cells containing the backbone vector (C127-IRES) and cells overexpressing full-length Polß (C127-Polß) revealed a dramatic alteration in the pattern of HinfI/RsaI fragments detectable with TTAGGG-repeat probe. Fig. 4ACitation shows that the TTAGGG-repeat fragments from C127-Polß cells (Lane 3) migrated between 9 and 1 kb (median, 5 kb), whereas from C127-IRES cells (Lane 2), they migrated between 5 kb and the bottom of the gel (median, 3 kb). By contrast, the TTAGGG fragments of primary MEFs from Polß–/– and Polß+/+ embryos migrated at 23 kb and showed no apparent changes (Lanes 5 and 6). Scanning and quantification of Lanes 3 and 4 of Fig. 4ACitation reveals that 45% of the TTAGGG signal obtained from cells ectopically expressing Polß is 4–8 kb in length, whereas only 30% of the signal from C127-IRES cells is within this range. This suggests that a larger fraction of the telomere population is between 4 and 8 kb in the C127-WT cells than in the control cells. The fact that Polß overexpression induced TTAGGG fragments that were larger than the size of the original telomeres suggested that these molecules might represent the chromosome end fusions that were first detected by cytogenetic analysis.



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  		Fig. 4. Polymerase ß (Polß) overexpression increases the telomere length. Genomic DNA was prepared from C127-internal ribosome entry site (IRES), C127-Polß cells, HeLa1.2.11 cells, Polß+/+ mouse embryonic fibroblasts (MEFs), and Polß–/– MEFs digested with HinfI and RsaI, separated by electrophoresis, transferred to a nylon membrane, and probed with an end-labeled telomere specific probe. The telomeres in cells overexpressing Polß (C127-Polß; Lane 3) appear to be longer than their control counterparts (C127-IRES; Lane 2). The HeLa subclone HeLa1.2.11 with long telomeres (23 kb; Lane 4) was used as a positive control. Telomeres from Polß+/+ MEFs (Lane 5) or Polß–/– MEFs (Lane 6) showed no apparent changes in their length. Molecular size markers consisting of 1 kb (Lane 1) and of {lambda}-bacteriophage HindIII restriction fragments (Lane 7) are shown.

 
In aggregate, the aforementioned results show that ectopic expression of Polß results in end-to-end fusions and increased telomere length in mouse C127-Polß cells, both of which are indicative of a role of Polß in telomere dysfunction. Telomere dysfunction occurs when telomeres are uncapped. Therefore, our data suggest that ectopic expression of Polß induces telomere uncapping or that uncapped telomeres resemble damaged DNA that could bind to DNA damage response factor(s) such as excess Polß.

Polß Associates with the Telomere-Binding Protein TRF2.
Telomere uncapping can occur when telomere-binding proteins no longer interact with the telomere. The results described previously could be explained if ectopic Polß disrupts the TRF2-dependent telomere protection. Therefore, we used immunoprecipitations to examine whether Polß and TRF2 interact physically. As Fig. 5ACitation shows, TRF2 was present in Polß immunoprecipitates in whole cell extracts made from the C127-IRES cells and the cells overexpressing the full-length protein (C127-Polß) (Lanes 1 and 4). In Polß-overexpressing cells, we observe two or three times more TRF2 coprecipitated with Polß (Lane 4) than in C127-IRES cells. We were unable to detect Polß in TRF2 immunoprecipitates (data not shown). It is possible that the TRF2 antibody interferes with this interaction or that the epitope recognized by the TRF2 antibody is blocked by Polß.



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  		Fig. 5. Polymerase ß (Polß) interacts with TRF2. A, TRF2 is present in Polß immunoprecipitates from C127 cells. Proteins were precipitated from C127-internal ribosome entry site (IRES; Lanes 1–3) and C127-Polß cell lysates (Lanes 4–6) by monoclonal antibodies (mAbs) Polß (Lanes 1 and 4) and TRF2 (Lanes 2 and 5). Immunoprecipitates then were subjected to SDS-PAGE electrophoresis on 10% gels, transferred to Immobilon-P, and probed with mAb TRF2 (1:10 dilution). Detection of bound mAb was with antimouse horseradish peroxidase (HRP)-conjugated secondary antibody at 1:10,000, followed by the enhanced chemiluminescence (ECL) system. B, Polß interaction with TRF2 is not DNA dependent. To exclude the possibility that the interaction is caused by DNA binding ability of both proteins, proteins were precipitated from 293T cell lysates alone (~1 mg/aliquot; Lane 1) or from cells transiently transfected either with the full-length hTRF2 fused to a GST-expression vector (Lanes 4 and 5) or the backbone vector alone (Lane 2) by mAb Polß in the absence (Lanes 1–3) or presence (Lanes 4 and 5) of DNase I. Immunoprecipitates then were subjected to SDS-PAGE electrophoresis on 10% gels, transferred to Immobilon-P, and probed with mAb TRF2. Detection of bound mAb was with antimouse HRP-conjugated secondary antibody at 1:10,000, followed by the ECL system. Whether DNase I treatment of the cell lysate was before (Lane 5) or after (Lane 6), the addition of primary antibody appeared not to affect the interaction (compare Lane 3 with Lanes 5 and 6). C, TRF2 interacts with the Mr 31,000 domain of Polß. Top, schematic representation of the domains of TRF2 and Polß proteins. Bottom, the deletion fragments of both proteins used in the yeast two-hybrid screening.

 
We addressed the possibility that the association of TRF2 with Polß was mediated by DNA tethering in 293T cells that were transiently transfected either with a full-length hTRF2 cloned into a GST expression vector (pEBGhTRF2) or with backbone vector. TRF2 again was present in Polß immunoprecipitates from each of the cell extracts, as shown in Fig. 5BCitation , Lanes 1–3, and more TRF2 coprecipitated with Polß in cells overexpressing TRF2. Moreover, DNase I treatment of the cell lysates before immunoprecipitation did not disrupt the interaction of TRF2 with the Polß (Fig. 5BCitation , compare Lane 3 with Lanes 5 and 6), indicating that the recovery of TRF2 in Polß immunocomplex is not likely to depend on the DNA binding activity of both proteins.

To corroborate these results, the yeast two-hybrid system also was used to map the interaction domains of TRF2 and Polß. Two fragments of the Polß gene were cloned into the bait vector, one that encodes the Mr 8,000 domain of Polß that contains the dRP lyase activity essential for repairing damage, and the other that encodes the Mr 31,000 domain that bears the polymerase activity (Fig. 5C)Citation . For TRF2, three fragments of 1 kb, 700 bp, and 500 bp, respectively, starting from the 3' end of the gene, were cloned into the prey vector (Fig. 5C)Citation . Subsequent transformation into yeast revealed that the Mr 31,000 domain of Polß mediated the interaction with TRF2 as assessed by ß-galactosidase activity. It also appears that the dimerization domain of TRF2 is required for the interaction with Polß (Fig. 5C)Citation because the shorter COOH-terminal TRF2 fragments are sufficient for the interaction.

Polß Associates with the Telomere-Binding Protein TRF2 to Nontelomeric Sites.
To examine the presence of Polß at telomeres, we used dual indirect immunofluorescence for Polß and TRF2 combined with telomeric fluorescence in situ hybridization (using an avidin-labeled CCCTAA DNA probe) in interphase nuclei. We chose to address this issue in interphase cells because the higher level of condensation of metaphase chromatin could result in misleading staining intensities. To detect Polß, we used a rabbit polyclonal IgG antibody that is specific for Polß (11) . Similarly, to detect TRF2, we used an mAb (IMG124; Imgenex). Using these antibodies, we examined the C127-IRES and C127-Polß cells in parallel, and the images were captured and reproduced under exactly the same conditions and settings.

Telomeric fluorescence in situ hybridization signals were randomly distributed across the entire nucleus in C127-IRES and C127-Polß cells as shown in Fig. 6, A and ECitation . In most interphase cells, TRF2 antibody showed the characteristic punctate TRF2 pattern throughout the nucleus consistent with previous reports. Notably, most of the TRF2 foci did not colocalize with the telomeric signals in either cell line (see arrows in Fig. 6, B and FCitation ). Polß immunostaining showed a similar punctate pattern of nuclear and cytoplasmic distribution in both types of cells (Fig. 6, C and G)Citation . Polß and TRF2 were colocalized in C127-IRES and C127-Polß cells only in some of the brightest foci (see arrowheads in Fig. 6, D and HCitation ). Although direct quantitative interpretation of immunofluorescence signals is not possible, colocalization of the two proteins was ~10–15% in C127-Polß cells and 5–8% in C127-IRES, as assessed by counting the numbers of foci that were merged in at least 50 cells. The colocalized signals of Polß and TRF2 never overlapped with the telomeric signals (see arrowheads in Fig. 6, D and HCitation ).



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  		Fig. 6. Polymerase ß (Polß) colocalizes with TRF2 to nontelomeric sites. Combined in situ hybridization and double indirect immunofluorescence staining for telomeric repeats (telo), Polß, and TRF2 in C127 cells overexpressing Polß (C127-Polß) or the backbone vector [C127-internal ribosome entry site (IRES)] and in four HeLa cell lines with the indicated telomere lengths. Indirect immunofluorescence signals for Polß and TRF2 proteins and in situ hybridization for the telomeric repeats (telo). Telomeres were detected before the immunofluorescence staining of the two proteins. The latter was executed in parallel, and images are presented without any adjustment. DNA was stained with 4,6-diamino-2-phenyllndole. A–D, telomeric signals, TRF2, and Polß staining in C127-IRES. E–H, telomeric signals, TRF2, and Polß staining in C127-Polß. I–L, telomeric signals, TRF2, and Polß staining in Hela1.2.11 cells. M–P, telomeric signals, TRF2, and Polß staining in HeLaS3. Q–T, telomeric signals, TRF2, and Polß staining in HeLa-hTERT cells. U–X, telomeric signals, TRF2, and Polß staining in HeLa-LXSN cells. Arrows indicate free telomeres, and arrowheads indicate TRF2 and Polß colocalization.

 
TRF2 signals were shown previously to be occasionally not associated with telomeric DNA (57) . TRF2 also has been shown to migrate to nontelomeric sites when it loses its DNA binding domain in an in vitro model (25) or in vivo when it associates with telomere-like sequences at a chromosome-internal site in certain hamster chromosomes (24 , 58) . The telomeric deficiencies observed in Polß-overexpressing mouse cells may be caused by their murine origin because it has been shown that mouse cells activate different damage signaling pathways to maintain telomeres compared with human cells (41) . Therefore, we examined the localization of TRF2 and Polß in human cells. We compared the TRF2 and Polß immunofluorescence signals in two pairs of closely related HeLa subclones that differ with regard to the length of the telomeric repeat tracts. Hela1.2.11 has telomeres of ~15–40 kb (24 , 32) , whereas HeLaS3 has shorter telomeres ranging from 5–10 kb (32) . The second pair were HeLa cells, which express a transduced hTERT gene (HeLa-hTERTf), that lengthen their telomeres up to 10 kb as opposed to their parental subclone (HeLa-LXSN), which has short telomeres of 3–5 kb (59) . All of the HeLa subclones express Polß as shown in Fig. 7Citation . Notably, however, the HeLa-LXSN cells with short telomeres express low amounts of Polß (Lane 4). Indirect immunofluorescence combined with in situ hybridization again was used to examine the abundance of Polß and TRF2 at interphase telomeres. The HeLa cells with long telomeres (Hela1.2.11 and HeLa-hTERT; Fig. 6, I and QCitation ) showed a pronounced punctate pattern of TRF2 with the majority of the signals localized at the telomeres in all of the interphase nuclei (Fig. 6, J and R)Citation . Interestingly, Polß shows a dispersed signal in cells with long telomeres (Fig. 6, K and S)Citation , which never colocalized with any TRF2 or telomeric signals in those cell lines (Fig. 6, L and T)Citation . Strikingly, in cells with short telomeres (HeLaS3 and HeLa-LXSN), Polß displays a punctate rather than a dispersed signal, which is not observed in cells with long telomeres. Telomeres and TRF2 signals were of reduced intensity and harder to detect in HeLa cells with short telomeres (HeLaS3 and HeLa-LXSN; Fig. 6, M, N, U, and VCitation ). Polß showed partial colocalization with TRF2 again only at nontelomeric sites (see arrowheads in Fig. 6, P and XCitation ).



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  		Fig. 7. HeLa subclones express different basal levels of polymerase ß (Polß) as assessed by Western blot analysis. Fifty µg of total protein made from HeLa1.2.11 (Lane 1), HeLaS3 cells (Lane 2), HeLa-hTERT (Lane 3), and HeLa-LXSN (Lane 4) were size fractionated by SDS-PAGE electrophoresis before blotting. Immunoblots were probed with an antitubulin monoclonal antibody (mAb; 1:3000 dilution) as an equal loading control and an anti-Polß mAb (1:500 dilution). Bound primary mAbs were detected with a sheep antimouse horseradish peroxidase-conjugated second layer in a 1:10,000 dilution and the enhanced chemiluminescence system.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The genetic and physical evidence presented demonstrates that excess Polß can induce chromosomal instability by affecting telomere metabolism. Chromosome end-to-end fusions accompanied by a change in the average telomere length were evident in cells overexpressing Polß. Moreover, Polß also was found to interact with the telomeric protein TRF2 in mouse and human cell lines. This finding was unexpected because Polß has been considered to be mainly a BER protein, whereas TRF2 is a telomeric protein. However, the physical interaction of Polß and TRF2 suggests that these proteins may function in a common DNA metabolic pathway.

Polß and Telomere Maintenance in Mouse Cells.
Primary MEFs deleted of the Polß gene do not appear to possess phenotypes associated with telomere dysfunction. We find that these early-passage MEFs have many strand breaks, which could eventually result from an inability of the Polß-deleted cells to fill gaps in the DNA during BER. However, the Polß-deleted primary MEFs have long telomeres, ~23 kb in length, and they are not fused. These observations suggest that Polß is not required for telomere maintenance during mouse development but that it is required for DNA repair. It is uncertain whether Polß is required to maintain telomeres in adults because mice that lack the Polß gene die just after birth; therefore, the status of their telomeres cannot be monitored. However, the interaction with TRF2 is consistent with the possibility that Polß could function in the protection of telomeres. The best-characterized functions of Polß are its dRP lyase and gap-filling activities during BER. Thus, Polß may function in BER at telomeres, being recruited to these sites by TRF2. If this were the case, one would expect Polß to be transiently associated with the telomere. Polß also may be recruited to the telomere by TRF2 to assist in the restructuring of t-loops so that they can be extended by telomerase. Polß is able to perform limited strand displacement synthesis, as it does during long-patch BER (60) . Thus, Polß may be able to remove or alter the structure of the D-loop, perhaps resulting in destabilization of the t-loop and permitting telomerase access to the telomere to extend it. Conversely, Polß could function in stabilizing the D-loop by limited strand displacement activity after telomerase has extended the telomere. Polß may carry out one of these functions in complex with other proteins such as WRN (61) . If Polß functions in the unfolding of the t-loop, it would most likely be transiently associated with this structure some time during S-phase, which could be difficult to detect by indirect immunofluorescence. It is formally possible that Polß could function at a nontelomeric site in a complex with TRF2 in the repair or unfolding of telomeres, but we consider this highly unlikely.

Polß and Telomere Dysfunction.
C127 cells have short telomeres, with a median length of 3 kb. In interphase cells, we were not able to detect a colocalization of TRF2 with the telomeric sequences. This indicates that when the telomeres are short in the C127 cells, TRF2 is unable to associate with the telomere, perhaps because the telomere is unable to form a t-loop. Alternatively, small amounts of TRF2 may be associated with some of the longer telomeres in the population, and this may not be detectable with standard immunofluorescence techniques. When we overexpress WT Polß in these cells through the use of a bicistronic vector, we observe end-to-end fusions and an increase in telomere length. These phenotypes have all of the hallmarks of telomere dysfunction. We suspect that the ability of Polß to interact with TRF2 plays an important role in eliciting these phenotypes. One possibility is that the chance for an interaction between Polß and TRF2 to take place increases in cells with greater amounts of Polß. This could result in the sequestration of TRF2 by Polß at nontelomeric sites, inhibiting its association with telomeres, which is consistent with the increased colocalization of Polß and TRF2 in the C127-Polß cells at nontelomeric sites. A second possibility is that TRF2 may give Polß access to the short telomeres in the C127 cells at times when it should not be at these sites. Once bound to the DNA, this polymerase could play a direct role in the fusion of telomeric ends, perhaps by functioning in concert with the nonhomologous end-joining machinery that is known to be important in the generation of end-to-end fusions (62) . This suggestion is consistent with our preliminary finding that cells expressing an inactive variant of Polß do not have large numbers of fused telomeres/cell and do not appear as giant cells in culture. A third possibility is that Polß interacts with other proteins in the cells, such as WRN, and sequesters these proteins away from the telomere, resulting in the phenotypes we observe (63) . Alternatively, Polß may act in a pathway that is independent of TRF2 to elicit the phenotypes we observe in the C127 cells.

Polß and Short Telomeres.
In the studies described here, we show that TRF2 associates with long telomeres in the HeLa1.211 and HeLa-hTERTf cells, whereas we are unable to detect association of TRF2 with short telomeres in the HeLaS3 and HeLa-LXSN cells during interphase. Interestingly, in the cells with short telomeres, we observe a focal pattern of Polß, but in cells with long telomeres, the Polß signal is diffuse. This suggests that the Polß foci form when cells have short telomeres. It is possible that cells with short, and perhaps uncapped, telomeres could signal a general DNA damage response, resulting in a focal pattern of Polß distribution. These foci may act to sequester Polß away from the telomere to ensure access of telomerase to the telomere or to prevent end-to-end fusions from taking place. The Polß foci also could represent sites of DNA repair, which may play a role in telomere maintenance.

In summary, we have shown that overexpression of Polß in C127 cells results in end-to-end fusions, which are a hallmark of telomere dysfunction. The fusions most likely arise as a result of the interaction of Polß with TRF2. At this point, it is not clear whether Polß plays a role in telomere maintenance, but too much Polß could induce genomic instability. Bergoglio et al. (19) recently found that overexpression of Polß induces genomic instability in Chinese hamster ovary cells, and this correlates with the ability of these cells to form tumors in mice. They demonstrated that Polß-overexpressing cells had a spindle point defect. On the basis of our current findings, we suggest that this could be because of fused telomeres in the Chinese hamster ovary cells. Telomeres also may accumulate mutations, especially in cells or tumors overexpressing Polß because Polß is known to be somewhat error prone. The accumulation of mutations at telomeres might result in the phenotypes we observed in our study because they could result in less binding of some of the sequence-specific telomere binding proteins, resulting in telomere instability.


   ACKNOWLEDGMENTS
 
We thank Ed Goodwin and Daniel DiMaio for the HeLa-LXSN and HeLa-hTERT cells; T. de Lange for the hTRF2 plasmid; Sheila Stewart, Ittai Ben-Porath, and Robert Weinberg for helpful advice regarding the telomere-oligonucleotide ligation assay; Mihai Ciubotariu for repeated helpful advice; and Mazin Qumsiyeh for advice in cytogenetics.


   FOOTNOTES
 
Grant support: NIH grants CA16038 and ES10995 (J. B. Sweasy). J. B. Sweasy is a Donaghue Investigator.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Joann B. Sweasy, Department of Therapeutic Radiology, 333 Cedar Street, P.O. Box 208040, New Haven, CT 06520. Phone: 203-737-2626; Fax: 203-785-6309; E-mail: joann.sweasy{at}yale.edu

Received 1/14/04. Revised 3/ 1/04. Accepted 3/25/04.


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 DISCUSSION
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