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Previous Article | Next Article 
Molecular and Cellular Biology, January 2008, p. 20-29, Vol. 28, No. 1
0270-7306/08/$08.00+0 doi:10.1128/MCB.01122-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
,
Cindy Groff-Vindman,2,
Sarah A. Compton,1
Michael J. McEachern,2 and
Jack D. Griffith1*
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,1 Department of Genetics, University of Georgia, Athens, Georgia 306022
Received 24 June 2007/ Returned for modification 20 July 2007/ Accepted 10 October 2007
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ABSTRACT |
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INTRODUCTION |
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Telomeres in the budding yeast Saccharomyces cerevisiae have been proposed to assume a fold-back structure mediated by protein-protein interactions, although the variable telomeric-repeat sequence may or may not allow strand invasion of the 3' overhang to generate a t-loop (10, 11). The Taz1 protein, from the fission yeast Schizosaccharomyces pombe, is an ortholog TRF2, and in vitro studies have shown that, like TRF2, it has the ability to arrange model telomere substrates into looped structures (32). However, direct examination of native yeast telomere architecture by electron microscopy (EM) to confirm the presence or absence of t-loops in yeast has proven problematic, due to the difficulty of isolating the short telomeres in these species. This is because long telomere restriction fragments (TRFs), which can be separated from the short genomic restriction fragments by gel filtration chromatography, are a technical requirement for observation of native telomeric DNA by EM.
While telomere attrition is most often counterbalanced by telomerase activity, homologous recombination-dependent telomere maintenance mechanisms also exist. A subset of human tumor and immortalized cells use a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT) that is believed to be dependent on recombination (16), and recombinational telomere elongation (RTE) is well characterized in yeast mutants with telomerase deleted (23). Such mutants display gradual telomere shortening, leading to senescence. However, survivors can emerge from senescence if chromosome ends have become elongated by recombination (20, 22). Two types of survivors are characterized in S. cerevisiae: type I has short telomeres and exhibits amplification of the subtelomeric Y' elements, while type II is similar to human ALT cells, exhibiting elongated, heterogeneous telomeric DNA (7, 20, 30). Survivors in the related budding yeast, Kluyveromyces lactis, are exclusively type II (22).
Current data support a model of type II RTE termed "roll-and-spread" in which extrachromosomal telomeric repeat (ECTR) DNA circles (t-circles), created by telomeric recombination, act as a template for rolling-circle replication, elongating one (or more) telomere (26). The elongated telomere(s) then spreads to other chromosome ends via nonreciprocal recombination events. Support for this model comes mainly from K. lactis, in which exogenous t-circles were shown to promote RTE and a DNA sequence from one elongated telomere was shown to spread to other telomeres following RTE onset (25, 26, 33). Evidence of a roll-and-spread mechanism was also observed in the mitochondria of the yeast species Candida parapsilosis, which contains linear mitochondrial DNA capped by telomeres maintained in a telomerase-independent fashion (28). Consistent with a role in RTE, t-circles are a conserved feature in human ALT cells (4, 36), S. cerevisiae survivors (18, 19), and C. parapsilosis mitochondria (31), and as described below, they likely reflect a t-loop origin. Recent data also indicate that Ku70 deletion in Arabidopsis thaliana results in t-circle formation and the growth of callus cultures with ALT-like telomeric phenotypes (39).
K. lactis provides an excellent opportunity for investigating telomere structure in a yeast species. Unlike S. cerevisiae, the 25-bp K. lactis telomeric repeat contains TA dinucleotides that allow interstrand cross-linking by psoralen (Fig. 1D), a necessary step for maintaining t-loop structure during isolation (14). K. lactis mutants with telomeres long enough to permit isolation from the bulk genomic DNA following restriction enzyme fragmentation are available, either with or without obvious telomere-capping defects (34). The ter1-16T mutant, in particular, is well suited for such studies. Mutation of the telomerase RNA gene in ter1-16T generates telomeric repeats with a base change that alters the binding site for Rap1, a protein crucial to telomere length regulation and capping (Fig. 1D) (34). The ter1-16T mutation leads to long, uncapped telomeres with abnormally long 3' overhangs, both of which can be suppressed by RAP1 overexpression (34). ter1-16T cells have also been shown to produce small t-circles using homologous recombination, and this can be inhibited by RAD52 deletion (15).
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cells contained significantly fewer t-loops and t-circles. Cumulatively, these data suggest that increased recombination at K. lactis telomeres in the absence of Rap1 results in increased strand invasion at t-loop junctions and resolution of t-loops by homologous recombination into free t-circles. |
MATERIALS AND METHODS |
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were constructed as described previously in a His-positive revertant of the 7B520 strain (34). The ter1-16T RAP1OE strain was generated by transforming ter1-16T with pCXJ3+RAP1 (a gift from Anat Krauskopf). ter1-16T and ter1-16T rad52 strains were transformed with pCXJ3 lacking a transgene. Transformants were selected on media lacking uracil and then grown in liquid yeast extract-peptone-dextrose medium containing 125 µg/ml G418, previously shown to maintain >100 copies of plasmid per cell (37). Psoralen cross-linking and genomic-DNA isolation and digestion. K. lactis cells were grown to an optical density at 600 nm of 15 (postexponential phase). One liter of cells was spheroplasted, and nuclear isolation was performed as described previously (17) using 100 µg/ml Zymolyase 100T (Seikagaku, Tokyo, Japan). Nuclei were treated with psoralen and UV light, and the genomic DNA was isolated as described previously (5, 14). Prior to restriction digestion, genomic DNA was incubated with Escherichia coli single-stranded DNA binding protein (SSB) to protect single-stranded DNA segments (5, 6) and then was digested for 2 h at 37°C with AluI, HpaII, and NlaIII (New England Biolabs [NEB], Beverly, MA) at 1 unit/µg of DNA each. These enzymes were chosen for their lack of single-stranded nuclease activity (NEB technical support). The mixture was supplemented with an equal amount of enzyme plus 20 µg/ml RNase A for an additional 2 h of incubation. Digestions were checked for completeness on a 1% agarose gel, and if required, more enzyme was added. The digested DNA was purified and prepared for gel filtration chromatography as previously described (5, 14).
Gel filtration chromatography. TRFs were fractionated as described previously (5). The telomeric contents of the eluted fractions were determined by slot blot analysis (5) using a 32P-labeled oligonucleotide K. lactis C-strand telomere probe and quantified using a Storm 840 PhosphorImager and associated software (Molecular Dynamics, Piscataway, NJ).
EM. DNA samples were prepared for EM by surface spreading on a denatured protein film (14). When DNA from the gel filtration column was subsequently digested with RsaI or MseI (NEB), the sample was brought to 0.1 µg/ml SSB in 1x NEB buffer 1, incubated on ice for 30 min, and then treated with 0.5 unit of restriction enzyme for 15 min at 37°C. EDTA was added to 5 mM, and then the mixture was passed through a G50 Sephadex (Pharmacia, New York, NY) spin column and prepared for EM by surface spreading. Staining of single-stranded DNA with SSB was done on ice for 30 min in 20 mM HEPES, pH 7.6, 50 mM NaCl, with 0.2 µg/ml SSB. The mixture was fixed with 0.6% glutaraldehyde for 5 min at room temperature and then directly mounted onto carbon foils for EM (13). All samples were examined in a Philips/FEI Tecnai 12 transmission electron microscope (Eindhoven, The Netherlands), and molecule measurements were done using Digital Micrograph software (Gatan, Pleasanton, CA). Statistical analyses were done using GraphPad software (GraphPad, San Diego, CA), and images were prepared for publication using Adobe Photoshop (Adobe, San Jose, CA).
Agarose gel electrophoresis.
DNA isolation and detection of single-stranded G-rich telomeric DNA by in-gel hybridization without denaturation were done as previously described (34). Measurement of TRF length by pulsed-field gel electrophoresis (PFGE) was done using aliquots from non-cross-linked preparations for future EM analysis, and the telomeric material was detected by denaturation and in-gel hybridization as described previously (5). For standard two-dimensional (2D) gel electrophoresis, restriction fragments were separated in the first dimension in 0.6% gold agarose (ISC Bio Express, Kaysville, UT) in 0.5x TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA) at 1 V/cm for 13.5 h. The gel was stained with ethidium bromide and visualized by UV light, and the appropriate lanes were excised. A 1.1% agarose (Invitrogen, Carlsbad, CA) gel in 0.5x TBE containing 300 ng/ml ethidium bromide was poured around the excised slab. Second-dimension electrophoresis was carried out in 0.5x TBE containing 300 ng/ml ethidium bromide at 6 V/cm for 3 h. The telomeric content was determined by in-gel hybridization or Southern blotting, following a denaturation step with a 32P-labeled oligonucleotide K. lactis C- or G-strand telomere probe. Circular-DNA markers were made by diluting HindIII-digested 
DNA to 10 ng/µl and ligating it overnight at 16°C with T4 DNA ligase. High-percentage agarose-chloroquine 2D gel electrophoresis and Southern blotting were done as previously described (15). All hybridizations were visualized using a PhosphorImager.
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RESULTS |
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, possess long, uncapped telomeres, with the latter strain not exhibiting telomere recombination and ECTR DNA due to the absence of RAD52 (34). Additionally, we used the RAP1-overexpressing strain ter1-16T RAP1OE, in which the telomere-uncapping phenotype is suppressed due to an abundance of Rap1p (34).
Undigested genomic DNA from ter1-16T, ter1-16T rad52, and ter1-16T RAP1OE cells was isolated, separated by standard gel electrophoresis, and subjected to in-gel hybridization under conditions that detected native single-stranded G-rich telomeric DNA (Fig. 1A). In ter1-16T and ter1-16T rad52, abundant single-stranded G-rich telomeric DNA was present, consistent with previous results (34) and indicative of an uncapped telomere state. We noted that ter1-16T rad52 showed a diminished amount of the small single-stranded G-rich material found in ter1-16T (Fig. 1A) (34). In the ter1-16T RAP1OE strain, the single-stranded G-rich telomeric signal was absent, indicating effective suppression of the uncapping phenotype (Fig. 1A).
Telomere length was assayed in all three strains by separation of uncut or AluI/HpaII/NlaIII (A/H/N)-digested genomic DNA using PFGE. A/H/N digestion of K. lactis genomic DNA leaves an average of 370 bp (estimated range, 145 to 450 bp) of subtelomeric DNA on the resulting telomere fragments. Consistent with previous results, ter1-16T contained elevated levels of ECTR DNA, exhibited by a smear of low-molecular-weight telomeric DNA between 5 kb and 0.5 kb in the uncut lanes (Fig. 1B), while the ter1-16T rad52 and ter1-16T RAP1OE strains lacked similar signals in the uncut material, indicating a reduction in ECTR DNA material. The ter1-16T and ter1-16T rad52 strains exhibited heterogeneous A/H/N-derived TRFs up to greater than 20 kb in length. A/H/N-derived TRFs from ter1-16T RAP1OE were shorter, with the bulk of the signal between 1.75 and 5 kb, which is still significantly greater than the 
300-bp telomeres in wild-type K. lactis.
Enrichment of TRFs from ter1-16T, ter1-16T rad52, and ter1-16T RAP1OE by gel filtration chromatography.
Observation of t-loop structures by EM is dependent upon first psoralen photo-cross-linking the nuclei, which is proposed to establish covalent bonds between the invading 3' overhang and the duplex telomeric DNA (8, 14). Crude nuclei from ter1-16T, ter1-16T rad52, and ter1-16T RAP1OE were psoralen photo-cross-linked, and the DNA was purified and reduced to TRFs by treatment with A/H/N. At least 0.5 mg of A/H/N-digested DNA from psoralen cross-linked or non-cross-linked samples from each of the strains was chromatographed over Bio-Gel A-15m. Elution profiles comparing the DNA concentrations and telomere contents indicated that for all preparations, the telomeric DNA preceded the bulk genomic DNA, providing the required enrichment for EM analysis (Fig. 1C).
Visualization of TRFs in ter1-16T and related strains. DNA from the high-molecular-weight, telomere-enriched fractions was prepared for EM. We quantified fractions starting at the peak of the telomere signal as determined by blotting and then examined earlier-eluting (higher-molecular-weight) fractions until the DNA concentration within the fractions became too low for EM analysis (Fig. 1C). Higher-numbered (lower-molecular-weight) fractions beyond the telomere peak contained increasing amounts of small DNA molecules, presumably representing the bulk genomic DNA. Tables 1 and 2 summarize the results for each preparation and statistical comparisons between conditions.
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ranged up to 30 kb, with an average size of approximately 7 kb, while fractions containing the peak telomeric signal had an average DNA molecule size of approximately 4 kb. In the telomere-enriched fractions from ter1-16T RAP1OE, the average DNA length was smaller, 2 to 4 kb, consistent with a decreased telomere length in this strain. As observed in previous studies of t-loops, linear DNA molecules were the most common structural form in the telomere-enriched fractions and are consistent with telomeres that were not arranged into a t-loop, telomeres in which strand invasions were not maintained during the enrichment procedure, or nontelomeric contaminants (4, 5, 14). Given that A/H/N digestion of whole K. lactis DNA is expected to produce only three nontelomeric restriction fragments of greater than 1,500 bp in size (genomic fragments of 1,787 and 1,731 bp and a 1,546-bp mitochondrial fragment), our observation of DNA fragments consistently larger than 2 kb suggests that the vast majority of the DNA molecules we observed were telomeric in nature.
Looped molecules are present in the psoralen cross-linked, telomere-enriched fractions from ter1-16T.
Lariat molecules consistent with the t-loop structure, described as a double-stranded DNA circle with a double-stranded DNA tail, were found in the telomere-enriched cross-linked fractions from ter1-16T (Fig. 2 and Table 1). A 3.6-fold-greater abundance in the percentage of looped molecules was observed in the cross-linked ter1-16T samples than in non-cross-linked samples, indicating a significant psoralen contribution to the stabilization of the loop structure (Fig. 3A and Tables 1 and 2). Additionally, when the cross-linked samples from ter1-16T RAP1OE and ter1-16T rad52 were compared to the cross-linked fractions from ter1-16T, they had significantly fewer looped molecules, 3.2- and 2.5-fold, respectively (Fig. 3A and Tables 1 and 2).
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for accurate size quantification. Looped molecules from ter1-16T are composed of telomeric DNA and are structurally similar to t-loops from higher eukaryotes. The 25-bp telomeric repeat in K. lactis contains a single RsaI site and no sites for MseI (Fig. 1D, MseI digests 5'-TTAA-3'). Incubation of multiple fractions from ter1-16T cross-linked preparation 2, with MseI, resulted in an increase in the abundance of looped molecules (Fig. 3C). Because these fractions correspond to the peak of the telomere signal in their respective elution profiles, we believe that this reflects the digestion by MseI of contaminating nontelomeric DNA in these late-eluting fractions, effecting a further purification step. In contrast, digestion of the same fractions with RsaI resulted in the complete disappearance of looped molecules and the almost complete reduction of the DNA within these fractions to sizes smaller than the limit of EM detection (Fig. 3C). The resistance of looped molecules from ter1-16T to MseI and their sensitivity to RsaI strongly suggests they are composed of telomeric DNA.
In the t-loop structure, a segment of single-stranded DNA is predicted to be present at the t-loop junction, due to strand invasion of the 3' overhang to generate the displacement loop (14). Consistent with this, E. coli SSB localized to the loop junction in 35% of t-loops isolated from human and mouse cells (14). We incubated DNA from the ter1-16T telomere-enriched, cross-linked fractions with E. coli SSB and directly mounted the samples onto carbon-coated EM grids. In these experiments, the SSB protein localized to the loop junctions of the majority of the looped molecules, suggestive of strand invasion-mediated telomere looping (Fig, 4A to C). The amounts of the SSB at the loop junction varied between a single SSB particle and large amounts of protein, suggesting considerable variability in the amount of single-stranded DNA present. This variability is likely due to heterogeneity in the elongated 3' overhangs in ter1-16T, which strand invade to form the displacement loop. Consistent with this, we also observed linear molecules from ter1-16T with variable amounts of SSB on one end (Fig. 4D and E).
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survivors is dependent on rolling-circle replication initiated on circular telomeric DNA as small as 100 bp (26). Previous examination of gel-isolated low-molecular-weight ECTR DNA from ter1-16T indicated that in this strain, small single-stranded and double-stranded t-circles were present, and that they ranged upward in size from 100 bp/nucleotide (15). In the telomere-enriched, cross-linked fractions from ter1-16T, we observed an abundance of circular DNA molecules (Fig. 5), ranging from 0.5 to 19.3 kb, with 84%, of the circles measuring <3 kb in size (n = 102; mean = 2.1 kb; median = 1.2 kb). DNA circles in psoralen cross-linked preparations from ter1-16T cells were significantly more abundant, 4.7- and 7.3-fold more prevalent on average, than in the psoralen cross-linked preparations from ter1-16T RAP1OE and ter1-16T rad52, respectively (Fig. 3D and Tables 1 and 2). Circular molecules in ter1-16T RAP1OE were also generally small, with 96% measuring <3 kb (n = 83; mean = 1.2 kb; median = 0.8 kb).
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(Fig. 6B and data not shown). In addition, we observed an arc of telomeric signal derived solely from the G-rich strand, with the bulk of the material migrating between 2 and 0.5 kb, which ran below the arc of linear double-stranded DNA in ter1-16T and ter1-16T rad52, but not ter1-16T RAP1OE (Fig. 6B). This is consistent with material in this arc being single-stranded G-rich ECTR DNA, suggesting that the increased single-stranded G-rich signal associated with telomere uncapping in budding yeast may be derived from both increased overhang length and ECTR DNA.
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Circular molecules were also quantified in the MseI and RsaI digestion experiments detailed above. In these experiments, RsaI digestion of telomere-enriched, cross-linked fractions from ter1-16T resulted in the complete disappearance of circles in one fraction and an 88% reduction in another, consistent with the circles containing telomeric DNA (Fig. 3E). MseI digestion also resulted in some reduction in circles (28% in one fraction and 21% in the other) (Fig. 3E), suggesting that treatment with MseI disrupted some t-circles or that some circles from ter1-16T observed by EM contained nontelomeric sequences.
The observation of RAD52-dependent t-circles in ter1-16T is consistent with a mechanism of circle formation due to homologous recombination resolution of the t-loop junction, resulting in a truncated telomere and a free t-circle (36). We therefore predicted a correlation in size between the loop portions of t-loops and t-circles, as observed previously in the human GM847 ALT cell line (4). In ter1-16T, there is good correlation between the sizes of t-circles and the loop portions of t-loops, with the greatest amount of discrepancy at the smaller size ranges (Fig. 3F). This likely reflects the loss of the smallest circular molecules during the gel filtration step.
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DISCUSSION |
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Several lines of evidence have led to the hypothesis that t-circles form from a recombination reaction involving t-loops. RAD52-dependent telomere rapid deletion (TRD) of overly elongated S. cerevisiae telomeres occurs via intramolecular recombination that is proposed to involve a t-loop intermediate (2, 21). Sudden telomere shortening in mammalian cells following TRF2B expression was dependent upon the recombination proteins XRCC3 and NBS1 and was accompanied by the formation of t-circles (36). t-circle formation in mice following POT1A deletion is also NBS1 dependent (38). In human ALT cells, t-circles have a size distribution similar to that of the loop portions of t-loops, suggesting they are derived from the same source (4), and t-circles were repressed in ALT cells following NBS1 and XRCC3 inhibition via RNA interference (9).
The data presented in this report strongly support the idea that t-circle formation in K. lactis is dependent on both recombination and t-loops. Not only are the frequencies of t-loops and t-circles in ter1-16T cells greatly diminished in the absence of RAD52, but the sizes of t-circles also correlate with the sizes of the loop portions of t-loops. Additionally, the small number of RAD52-independent t-loops and t-circles we observed could also be related to TRD, as a subset of TRD events were previously shown not to require RAD52 (2). One implication of our link between t-loops and t-circles is that both the formation and the resolution of t-loops are likely to be critical steps in any RTE that is driven by t-circles. A second implication is that t-loops occur in any of the budding yeasts or mutants in which t-circles have been observed (18, 19). As the resolution of t-loop junctions by homologous recombination is predicted to generate t-circles that have a nick or gap on both strands, this may explain why psoralen appears to stabilize t-circles in our preparations, as well as the exclusively open circular form of t-circles observed in this study.
When RAP1 is overexpressed in ter1-16T cells, there is a sharp decrease in the incidence of both t-loops and t-circles. This, however, does not indicate that t-loops are solely associated with an uncapped-telomere phenotype in K. lactis, as the very long overhangs present at ter1-16T telomeres are expected to greatly improve the efficiency of psoralen cross-linking within the t-loop junction. Therefore, even if the short overhangs of wild-type and ter1-16T RAP1OE K. lactis cells are sequestered within t-loops, our isolation protocol might be poorly able to cross-link these structures and recover them intact. Thus, while we cannot assign an architecture to wild-type K. lactis telomeres, we can conclude that in ter1-16T RAP1OE the telomeres are structurally arranged in a way that prohibits excessive strand invasion at a t-loop junction, leading to a diminished recovery of t-loops or t-circles. We propose that poor occupancy of Rap1 protein at the mutant telomeric repeats present in ter1-16T cells leads to an altered telomere structure that renders the telomeres more recombinogenic, including a greater amount of strand invasion at the t-loop junction (Fig. 7). Strand invasion of a telomeric end under the recombinogenic state of telomeres lacking Rap1 would be prone to some or all of the processes that might act on a strand-invaded broken DNA end that has formed a D-loop, including branch migration at the t-loop junction, nucleolytic cleavage resulting in TRD and a free t-circle, or extension by a DNA polymerase.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health to J.D.G. (GM31819 and ES 013773) and to M.J.M. (GM61645). C.G.-V. was supported through a training grant from the National Institutes of Health (5T32GMOO&10330) and S.A.C. though a National Institutes of Heath postdoctoral training grant (GM077900-01).
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FOOTNOTES |
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Published ahead of print on 29 October 2007.
Present address: Children's Medical Research Institute, Westmead, New South Wales 2150, Australia.
A.J.C. and C.G.-V. shared first authorship.
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0.5 ng/µl DNA) were bound to a nylon membrane and probed with the [32P]-C-tel probe. The signal was quantified using a PhosphorImager and plotted as relative telomeric intensity. Elution profiles from cross-linked samples are shown here. (D) Telomere sequence of ter1-16T. The RsaI (5'-GTAC-3') and Rap1 binding sites are indicated. * marks the G-to-T change at position 16, and # indicates proposed psoralen cross-linking sites at TA dinucleotides.
