Telomere Loops and Homologous Recombination-Dependent Telomeric Circles in a Kluyveromyces lactis Telomere Mutant Strain

Telomeres are the nucleoprotein structures at eukaryotic chromosome termini that cap the chromosome ends. They consist of tandem G-rich repeats that terminate in a single-stranded 3′ overhang and proteins that interact with both the single-stranded and double-stranded regions of telomeric DNA. Disruption of telomere capping can lead to one or more outcomes, including telomere fusions, increased telomeric recombination, and replicative senescence (12). In higher eukaryotes, telomeres are commonly arranged into a structure termed a telomere loop (t-loop) that involves strand invasion of the 3′ overhang into internal telomeric repeats of the same chromosome end (14). Such loops have been seen in human, murine, chicken, plant, and ciliate telomeres (5, 14, 24, 27). T-loop formation in vitro is facilitated by human TRF2 protein (1, 14, 29), and inhibition of TRF2 in vivo results in telomere dysfunction and chromosome fusions (3, 35), suggesting that the ability to stabilize t-loops may be an important part of telomeric capping. 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. ​(Fig.1D),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. ​(Fig.1D)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). FIG. 1. Phenotypic characteristics and enrichment of telomeric DNA from K. lactis strains. (A) Detection of single-stranded G-rich telomeric DNA by in-gel hybridization without denaturation. Uncut genomic DNA (1 μg) was separated on a 0.8% agarose ... In this study, we have isolated telomeric DNA from the K. lactis ter1-16T strain for direct examination by EM and 2D gel analysis. We show that telomeric DNA from ter1-16T cells contains looped molecules of telomeric DNA with the physical characteristics of t-loops previously isolated from mammalian and plant cells, as well as abundant t-circles ranging up to the size of an entire telomere. In contrast, telomeric DNA from ter1-16T RAP1OE (overexpressing RAP1) and ter1-16T rad52Δ 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.