Recombination Can Cause Telomere Elongations as Well as Truncations Deep within Telomeres in Wild-Type Kluyveromyces lactis Cells

Telomeres are complexes of DNA and proteins that protect the ends of chromosomes from being recognized as double-strand breaks (9, 28, 50). Due to the inability of replicative polymerases to fully replicate ends, telomeres gradually shorten over time in the absence of a specialized means of maintaining them (48, 75). In the majority of eukaryotes, the ribonucleoprotein telomerase acts to extend telomere ends by adding nucleotides to a short 3′ overhang at telomeres (49, 63). In what is thought to be an anticancer adaptation, most human cells have little or no telomerase, which leads to replicative senescence when telomeres become too short (62). Furthermore, telomeres are normally “capped” by a complex of proteins which protect them from nonhomologous end joining (NHEJ) and homologous recombination (11, 51). In some organisms, including humans, telomere capping may also involve telomeric loops (t-loops), the structures resulting from the intramolecular strand invasion of the 3′ overhang at telomeres into the internal telomeric repeats (14, 37). While gradual loss and extension by telomerase are the most common modes of telomere length changes, other processes, most notably recombination, have been shown to affect telomere length under certain circumstances. Some DNAs, including the chromosomes of the mosquito Anopheles gambiae and the linear mitochondrial DNAs of certain yeasts and ciliates, appear to use recombination as their normal method of telomere maintenance (36, 47, 59, 66). Telomerase-deficient mutants of a number of yeast species are able to maintain their telomeres by recombination (27, 30, 42, 64). In Kluyveromyces lactis and Saccharomyces cerevisiae, the species that have been studied the most, telomere elongation appears to occur by a “roll-and-spread” model, in which a small circle of telomeric repeats (t-circle) first acts as a template for rolling circle DNA synthesis to elongate a single telomere. The sequence from the first long telomere can then be copied in a break-induced replication (BIR)-like event to elongate the other telomeres in the cell (15, 26, 43, 44, 67). In both species, this recombinational telomere elongation is dependent on the major recombination protein encoded by RAD52 and is initiated by the loss of telomere capping brought on by very short telomere lengths (27, 30, 33). More recent work has demonstrated that recombinational telomere elongation can also occur as a consequence of certain mutations directly affecting telomere capping (2, 12, 13, 18, 22, 53, 78). Interestingly, these mutations frequently create telomeres that are longer and more heterogeneous than telomeres undergoing recombination caused by the loss of telomerase. Recombination has also been shown to be an important telomere maintenance mechanism in some cancer cells. Telomerase activity is very low or absent in most human somatic cells (20). While the majority of human cancers reactivate telomerase to maintain telomeres and immortalize cells, a significant minority use a recombinational mechanism called alternative lengthening of telomeres (ALT) (3, 20, 58). Telomeres become highly heterogeneous in ALT cancer cells, with some telomeres being very long and others having few or no telomeric repeats. Extrachromosomal telomeric DNA, in both linear and circular forms, is commonly found in these cells (6, 41, 74). Various types of telomeric recombination have been demonstrated to be common in ALT cells. Experiments using a sequence tag introduced on one telomere have shown that recombination events can transfer the sequence to other telomeres as well as duplicate it at the same telomere (10, 38). Also, reciprocal sister chromatid exchanges have been shown to occur at greatly elevated rates in ALT cells (1). Telomeres can also be subject to dramatic shortening events under a variety of different circumstances, including normal growth and development in some organisms. Examples include the abrupt shortening of all telomeres in newly developed macronuclei of the ciliate Euplotes crassus (71), the rapid trimming back to normal size of telomeres greatly lengthened by continuous mitotic growth in Tetrahymena thermophila (23), and the large truncations and elongations occurring during antigenic variation in Trypanosoma brucei (40). Even the comparatively large telomere attrition per cell division of relatively long telomeres in human fibroblasts and in telomerase mutants of Caenorhabditis elegans has been proposed to involve additional mechanisms besides gradual loss due to the end-replication problem (7, 17). Considerable evidence now suggests that oxidative damage to telomeres can accelerate telomere attrition in human cells (57, 61, 72, 73). Sudden shortening events are commonly associated with telomeres that have capping defects. Early studies of immortalized human cells showed dramatic shortenings, sometimes losing all telomeric signal, at a single telomere that had become long (39). Human cells containing mutations of the double-stranded telomere binding protein TRF2 showed a depletion of 3′ overhangs and telomere deletions that were the size of t-loops (70, 74). A K. lactis mutant with very long telomeres occasionally displayed better-growing colonies with much shorter telomeres (31). Even in situations where recombinational maintenance is occurring, there can also be concurrent large shortening events. Evidence from a K. lactis mutant lacking telomerase and having telomeric repeats defective for binding of Rap1 suggested that rapid truncations to very short lengths occurred regularly in cells with very long telomeres maintained by recombination (2). The best-studied example of dramatic telomere shortening is telomere rapid deletion (TRD), which has been shown to truncate abnormally long telomeres in S. cerevisiae (25). These truncations predominantly shorten telomeres down to the size of resident wild-type telomeres and were proposed to represent a trimming mechanism that shortened telomeres that had become too long. TRD in S. cerevisiae is partially dependent on the major recombination protein Rad52 and also shows a dependency on the Mre11p-Rad50p-Xrs2p complex and the Ku70/80 heterodimer (4, 25). The retention of introduced HaeIII sites at relatively more basal positions in telomeres that had undergone TRD led to the proposal that TRD occurred though a terminal deletion initiated by a t-loop intermediate (4). TRD showed a 30- to 70-fold increase in meiosis, which was dependent on the meiotic bouquet protein Ndj1p (19). TRD has also been reported for abnormally long telomeres in Arabidopsis thaliana but does not appear to be dependent on paralogs of RAD51 or on the MRE11-RAD50-NBS1 complex (76). Interestingly, in human cells overexpressing telomerase RNA, telomeres become long and heterogeneous in length and t-circles become abundant, the latter presumably because of trimming by a TRD-like mechanism (54). In order to determine the fate of a long telomere in K. lactis cells, we introduced an abnormally long telomere made up of phenotypically silent repeats containing a BclI restriction site into cells with otherwise wild-type telomeres. Like the case for S. cerevisiae, we observed abundant TRD events that shortened telomeres to apparent wild-type size. However, about half of these events were associated with deep turnover of telomeric repeats. We also report a lower frequency of further elongation of the Bcl telomere brought about by recombination.