Oncogene (2002) 21, 522 ± 531 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Telomere maintenance without telomerase Victoria Lundblad*,1 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, TX 77030, USA Recombination-dependent maintenance of telomeres, ®rst possibility that a potentially similar pathway operating discovered in budding yeast, has revealed an alternative in human cells, referred to as ALT (for Alternative pathway for telomere maintenance that does not require Telomere Lengthening), might contribute to neoplastic the enzyme telomerase. Experiments conducted in two transformation. This review discusses the investigations budding yeasts, S. cerevisiae and K. lactis, have shown in budding yeast that have helped elucidate this recombination can replenish terminal G-rich telomeric recombination-dependent process, followed by a dis- tracts that would otherwise shorten in the absence of cussion of the possible mechanisms that have been telomerase, as well as disperse and amplify sub-telomeric proposed as a result of the yeast studies. A brief repeat elements. Investigation of the genetic requirements summary of the ALT pathway, and a discussion about for this process have revealed that at least two dierent whether ALT proceeds through a similar recombina- recombination pathways, de®ned by RAD50 and tion-dependent pathway, concludes this review. The RAD51, can promote telomere maintenance. Although reader is also referred to other excellent reviews on this critically short telomeres are very recombinogenic, topic (Biessmann and Mason, 1997; Kass-Eisler and recombination among telomeres that have only partially Greider, 2000; Reddel et al., 2001; Kraus et al., 2001). shortened in the absence of telomerase can also contribute to telomerase-independent survival. These observations provide new insights into the mechanism(s) Discovery of a telomerase-independent pathway for by which recombination can restore telomere function in telomere maintenance yeast, and suggest future experiments for the investiga- tion of potentially similar pathways in human cells. Several observations during the 1980's provided early Oncogene (2002) 21, 522 ± 531. DOI: 10.1038/sj/onc/ clues that recombination could potentially maintain 1205079 telomeric repeats at chromosome ends. Linear plasmids that lacked a functional telomere, but contained Keywords: telomerase; EST1; ALT; recombination; homology to sub-telomeric regions, could acquire a break-induced replication functional chromosome terminus in a homology- dependent process (Dunn et al., 1984). Rescue of these linear plasmids was proposed to involve strand Linear chromosomes must have a mechanism to ensure invasion of the sub-telomeric region of a chromosome that their termini are fully duplicated when the by the terminus of the linear plasmid, followed by chromosome itself is replicated. For most eukaryotes, replicative copying of the donor onto the end of the the ends of nuclear chromosomes are maintained by plasmid (Figure 1a). The concept that a genetic the reverse transcriptase telomerase. Telomerase adds recombination intermediate could be converted into a species-speci®c telomeric repeats onto chromosome replication fork had already been proposed as a termini, thereby counterbalancing sequence loss that mechanism for replication of the ends of the linear would ensue as a result of semi-conservative DNA bacteriophage T4 genome (Luder and Mosig, 1982). replication by the conventional DNA replication Formosa and Alberts (1986) provided biochemical machinery (for reviews, see Greider, 1996; McEachern support for the contribution of recombination proteins et al., 2000). This not only ensures complete replication to the formation of a replication fork, and further of the genome, but the telomeric repeats that are suggested that homology-initiated DNA replication synthesized by telomerase provide a binding site for the might also apply to the telomere end replication host of telomere-speci®c proteins that prevent recogni- problem. Similar recombination-based models for tion of chromosome termini as double strand breaks telomere maintenance had also been elaborated by (for reviews, see Lundblad, 2000; Blackburn, 2001). Bernards et al. (1983) and Walmsley et al. (1984). However, increasing interest has been focused on a Direct demonstration that recombination could main- recombination-dependent mechanism for replenishing tain the natural termini of linear chromosomes, however, telomeric repeats. This pathway, ®rst discovered in awaited the analysis of telomerase-defective yeast budding yeast, is receiving attention due to the strains. The discovery of the S. cerevisiae EST1 gene (Lundblad and Szostak, 1989), later shown to encode a subunit of the telomerase holoenzyme (Hughes et al., *Correspondence: V Lundblad; E-mail: [email protected] 2000), allowed examination of the consequences of a Telomere maintenance without telomerase V Lundblad 523 essential genes (as a result of extensive terminal deletions) and/or chromosome transmission failures resulting from end-to-end fusions that trigger a break- age-fusion-bridge cycle. linear plasmid critically short telomere Although the majority of yeast cells die in the absence resection resection of telomerase, Lundblad and Blackburn (1993) observed that a small subpopulation could escape the lethal consequences of a telomerase defect. The rare survivors 3’ 3’ recovered from an est1-D strain not only regained the ability to grow but also displayed dramatic changes at strand exchange strand exchange their chromosomal termini, due to global ampli®cation of both telomeric and sub-telomeric repeat sequences. These extensive rearrangements arose as a result of recombination, as the appearance of survivors was extension of extension of invading strand invading strand blocked if the est1-D strain was also defective for RAD52, which is responsible for the majority of homologous recombination events in yeast. Survivors could be grouped into two general classes based on the second strand second strand pattern of ampli®cation of telomeric and/or sub- synthesis synthesis telomeric sequences. Although survivors were recovered as healthy growing clones arising from a culture of mostly inviable cells, continued single colony propaga- tion of both classes of survivors revealed variable growth final products final products patterns: sub-clones could be isolated in which a senescence phenotype re-appeared, although survivors readily reappeared again from these senescing sub- clones. Highly dynamic changes in telomere structure, linear healed plasmid with sequences rapidly lost or acquired at individual Figure 1 Two alternative mechanisms by which break-induced telomeres of survivor strains, was also observed, replication can capture a telomere. (a) A linear plasmid that providing a possible explanation for the growth contains homology to the sub-telomeric Y' repeat can initiate variability. Finally, neither est17 strains early in strand invasion at a chromosomal Y' element. Subsequent senescence nor subsequently isolated survivors displayed replication results in the nonreciprocal translocation of intact Y' and terminal G-rich telomeric repeats onto the end of the linear a genome-wide hyper-recombination phenotype. How- plasmid. (b) Strand invasion to initiate break-induced replication ever, this analysis did not exclude the possibility that in a can occur between a critically short telomere that still retains cell that lacked telomerase, unreplicated telomeres could telomeric repeat sequences and another terminus, thereby themselves become hyper-recombinogenic, a point that elongating the shortened telomere. Staggered alignment of the invading and donor strands (not depicted here) could also result will be re-addressed later in this review. in termini that are longer than chromosome ends maintained by Although all survivors recovered from an est1-D telomerase strain shared common features, such as RAD52- dependence and highly dynamic changes at their telomeres, they could be grouped into two classes telomere replication defect in an easily experimentally based on notable dierences in their telomeric manipulated organism. A haploid yeast strain deleted for structure. One class of survivors was distinguished by the est1-D gene exhibited gradual telomere shortening extensive ampli®cation of sub-telomeric repeats (called and an accompanying progressive decline in viability, Y' elements). In most wild type strains of S. cerevisiae, dubbed yeast cellular senescence. This established an approximately 2/3 of the telomeres contain tandem experimental paradigm for the relationship between arrays of one to four Y' elements, separated by short telomere replication and limits on replicative capacity, stretches of *50 to 100 bp of G-rich telomeric repeats which was recapitulated in human cells several years ago (Figure 2a). In a telomerase-pro®cient strain, exchanges (Bodnar et al., 1998). In addition, the rate of loss of a of Y' elements between telomeres had been shown to non-essential reporter chromosome increased, in parallel occur at low frequency, resulting in the occasional with the appearance of est17 senescence (Lundblad and dispersal of these repeats among chromosome ends Szostak, 1989). Since chromosome loss is the most (Horowitz and Haber, 1985). However, in one class of probable outcome of a DNA double strand break survivors, Y' copy number increased dramatically (Kramer and Haber,