Recombination at long mutant telomeres produces tiny single- and double-stranded telomeric circles.

Telomeres are the nucleoprotein structures found at the ends of linear chromosomes (2, 6, 9). Telomeric DNA consists of tandem arrays of short repeats that have G-rich and C-rich strands. The telomeric DNA is divided into a proximal double-stranded region and a more distal single-stranded 3′ overhang (36). This overhang is comprised solely of the G-rich strand and is elongated through the activity of the reverse transcriptase telomerase (53). Telomerase minimally consists of a template RNA that is reverse transcribed onto telomeric ends by the catalytic protein subunit. Telomeric DNA also associates with a large assemblage of proteins (43, 51). The telomere protein-DNA complex acts as a cap that is dynamic in composition and arrangement. It is the dynamic protein-DNA telomere complex that both controls telomere metabolism and protects telomeres from being recognized as double-stranded breaks. One proposed mechanism for telomere protection is the creation of telomeric secondary structures, such as t-loops, which are stabilized through the action of telomeric DNA binding proteins (19, 49). The maintenance of telomere length is important to the normal functioning of cells. In most human somatic cells, telomerase activity is very low or absent, so that telomeres gradually shorten, eventually reaching a state which triggers a growth arrest called replicative senescence (15, 40). Mutations or other conditions that bypass senescence produce further telomere shortening and take cells into crisis, a state of high genetic instability and cell death caused by widespread telomere dysfunction (41). In order to survive crisis and become immortal, a means of telomere elongation must arise (14, 39). Most cancer cells are immortal due to the presence of telomerase (40). A subset of cells that emerge from crisis maintain their telomeres through alternate lengthening of telomeres (ALT), a process that involves intertelomeric recombination (3, 16, 38). Studies have shown that telomeres of ALT cells are both highly heterogeneous in size and frequently much longer than telomeres in normal human cells and telomerase-positive cancer cells (3, 4). ALT cells also commonly contain intranuclear structures known as ALT-associated PML bodies, or APBs. APBs contain recombination proteins, telomeric DNA, and replication factor A and have therefore been suggested to play a role in the mechanism of ALT telomere elongation (33, 55). While the structure of telomeric DNA associated with APBs is unknown, extrachromosomal telomeric repeats (ECTR) have recently been extracted from ALT cell lines and have been shown to at least partially exist in circular conformations (7, 52). Yeast cells have constitutively active telomerase and normally maintain their telomeres within a fixed size range (17). However, deletion of telomerase in yeast cells leads to telomere shortening and coincident growth senescence (27, 29, 42). Although most cells eventually die from this senescence, some postsenescence survivors emerge. These survivors, which are dependent upon the recombination gene RAD52 (26, 28), elongate their telomeres through recombinational telomere elongation (RTE), a process that appears to be analogous to human ALT. In Saccharomyces cerevisiae, there are two pathways of RTE, each of which produces a distinctly different elongation pattern. Type I survivors have amplification of the long Y′ subtelomeric elements and short extensions of the telomeric TG(1-3) repeats (10, 26, 46). Type II survivors lack subtelomeric amplification and are characterized by long telomeric TG(1-3) extensions which are heterogeneous in length (10, 46). Both type I and type II survivors of Saccharomyces require RAD52 as well as type-specific sets of recombination genes. Type I survivors require RAD51, RAD54, and RAD57, while type II survivors require components of the MRX complex, RAD59, and the helicase SGS1 (10, 12, 21, 24, 45). Only type II survivors arise when the telomerase RNA gene (TER1) is deleted from Kluveromyces lactis cells (28). Inspection of telomeric sequence from K. lactis ter1-Δ survivors derived from cells with telomeres composed of basal wild-type and terminal phenotypically silent mutant repeats revealed repeating patterns containing both types of repeats (35). Most or all telomeres within a clone shared a single pattern, but patterns varied between survivor clones. These data led us to propose the roll-and-spread model, which proposes that the first elongated telomere is made within a cell by rolling circle replication around a circle as small as 100 bp and that the sequence of this long telomere is then spread by gene conversion to most or all other telomeres (35). Consistent with this model, K. lactis cells can utilize transformed telomeric circles of 1.6 kb and 100 nucleotides (nt) to elongate their telomeres (34, 35). Other recent work has confirmed that the sequence of one elongated telomere spreads at very high efficiency to all other telomeric ends during survivor formation (49a). In this work we show that the unusually small telomeric circles proposed by the roll-and-spread model can be made by recombination in a long telomere mutant of K. lactis.