Eukaryotic cells, including some human cancers, that lack telomerase can sometimes maintain telomeres by using recombination. It was recently proposed that recombinational telomere elongation (RTE) in a telomerase-deletion mutant of the yeast Kluyveromyces lactis occurs through a roll-and-spread mechanism as described in our previous work. According to this model, a tiny circle of telomeric DNA is copied by a rolling-circle mechanism to generate one long telomere, the sequence of which is then spread to all other telomeres by gene-conversion events. In support of this model, we demonstrate here that RTE in K. lactis occurs by amplification of a sequence originating from a single telomere. When a mutationally tagged telomere is of normal length, its sequence is spread to all other telomeres at a frequency (Ϸ10%) consistent with random selection among the 12 telomeres in the cell. However, when the mutationally tagged telomere is considerably longer than other telomeres, cellular senescence is partially suppressed, and the sequence of the tagged telomere is spread to all other telomeres in >90% of cells. Strikingly, the transition between a state resistant to recombination and a state capable of initiating recombination is abrupt, typically occurring when telomeres are Ϸ3-4 repeats long. Last, we show that mutant repeats that are defective at regulating telomerase are also defective at regulating telomere length during RTE.alternative lengthening of telomeres ͉ rolling circle

Telomeres are the specialized structures at the ends of eukaryotic chromosomes and are composed of short T/G-rich DNA repeats and the proteins that interact with them. Internal to telomeres are subtelomeric regions that are species-specific and often repetitive. The yeast Kluyveromyces lactis has telomeric tracts of 10-20 copies of a 25 bp repeat, but the subtelomeric regions have not previously been characterized in detail. Here we have cloned and characterized subtelomeric regions from 10 of the 12 chromosome ends. The amount of sequence examined was 0.7-10 kb for each subtelomeric region. We have identified a K. lactis subtelomeric element, the R element, which has a strong purine/pyrimidine strand bias and extends for about 2 kb. Internal to the R element, we found extensive similarity that is shared among half of the chromosome ends reported here. This similarity appears to include three putative gene families, two of which are also subtelomeric in Saccharomyces cerevisiae.

Both subtelomeric and telomeric recombination events can be greatly enhanced in Kluyveromyces lactis mutants lacking telomerase and having abnormally short telomeres. In this study, we utilized cells containing a single telomere composed of mutant repeats carrying a phenotypically silent mutation to test whether the exchange of telomeric repeats was a frequent event in mitotic and meiotic wild-type K. lactis cells. Amongst more than 100 subclones followed during multiple passages of mitotic growth, one instance of a terminal duplication extending into a subtelomeric sequence was observed, but no occurrences of intertelomeric recombination were found. This suggests that intertelomeric recombination is not an important contributor to telomere maintenance in normal K. lactis cells. Rare recombination events resulting in the replacement of a subtelomeric marker with a sequence from another chromosome end also led to the replacement of the telomeric repeat tract. This is consistent with these events being a result of break-induced replication. Movement of a subtelomeric or telomeric sequence from one chromosome end to another was not observed in haploid cells derived from mating and sporulation. This suggests that the exchange of telomeric repeats is not a routine occurrence in K. lactis meiosis.