Background

The Basic Structure of the Yeast Telosome. The yeast telomere nucleates numerous multimeric complexes. The double-stranded portion of telomeric TG1-3 tracts is bound to multiple copies of the major telomere binding protein Rap1. Rap1 forms two major complexes; one associated with the negative regulators of telomerase Rif1 and Rif2[1], [2], [3] and a second associated with the heterochromatic proteins Sir3p and Sir4 (Figure 1; [4]). In addition, the Ku 70/Ku80 heterodimer associates with the terminus where it associates with the RNA component of telomerase[5] and is critical for telomere size control, telomere cap function, and specific positioning of telomeres to the nuclear periphery ([6],[7],[8],[9],[10],[11]). An additional complex, MRX (Mre11/Rad50/Xrs2), is also important for telomere 3’ end processing and telomerase recruitment ([12], [13]).

Figure 1. The Telomere Equilibrium. The telomere is shown adjacent to the Y’ and X subtelomeric elements in an equilibrium between association of silencing and negative regulators of telomere size.

The Maintenance of Telomere Size: There are three basic components of telomere size control: the telomerase holoenzyme, complementary strand synthesis, and the maintenance of a mean telomere size at a genetically controlled constant value.

(a) Telomerase Synthesis---Most eukaryotic telomeric DNA is composed of simple G + T-rich sequence, following the consensus {(T/A)1-4N0-4G1-8}, with the G-rich strand oriented in a 5' to 3' direction towards the terminus ([14], [15]). In yeast, this sequence is composed of a TG1-3 irregular repeat. The catalytic reverse transcriptase subunit (Est2p in yeast) ([16],[17]) and the telomerase RNA (TLC1 in yeast) ([14],[17]) are sufficient for activity in vitro and define the "core" enzyme. TLC1 acts as a template for the addition of telomeric repeats onto G-rich single-stranded substrates both in vivo and in vitro ([18],[19],[20],[21],[22],[23]). Telomerase activity is utilized for telomere addition in the vast majority of eukaryotes ([15]).

The Telomerase Mechanism for Telomere Addition-Additional components of the telomerase RNP holoenzyme are essential for activity in vivo. One class of mutations defines genes that encode non-catalytic components of the holotelomerase. (e.g., Est1, Est3p, and Cdc13/Est4p) ([24], [25]). First, Cdc13 bound to the elongated single-stranded overhang in late S phase may selectively recruit Est1, which, in turn, recruits telomerase ([25], [26] ([13]). A second set of studies suggested a cell-cycle and Cdc13-independent binding of the core enzyme throughout the cell cycle with S-phase specific activation of telomerase, possibly mediated through interactions with Cdc13 and Est1 ([27], [28]). The complementary strand is synthesized in coordination with telomerase by components of the conventional lagging strand synthesis machinery, including Pol d Pol a, and RNA primase ([29],[30],[31]) coupled with a nucleolytic mechanism for generating a new 3’ end ([32], [32],[33, 34],[35]).

Alternative Mechanisms for Telomere Elongation- In telomerase-negative cells or in cells containing aberrant telomere structures (e.g., short telomeres, unprocessed telomeric ends or overelongated 3’ overhangs), recombination pathways allow the survival of the population. Two predominant types of pathways have been identified. Type I recombination amplifies subtelomeric Y’ elements ([36], [37],[38],[39]) and is dependent on both Rad52 and Rad51, the major yeast RecA strand exchange homologue. The underlying mechanism of Type I recombination may involve gene conversion through either single strand annealing or strand invasion in G2/M arrested cells during senescence ([40]).

However, a more efficient pathway for survival is RAD51-independent RAD52-dependent Type II recombination between telomeric sequences ([36],[37],[38],[39]). Type II recombination may be initiated by continual cycles of replication of either an extra-chromosomal excised circle (rolling circle) or by replication through an intrachromatid t-loop intermediate (‘rolling loop’ see Figure 2) ([41],[42],[39, 43]).

This process is triggered by shortened or improperly processed termini ([44]). Type II recombination is also dependent on Rad59, a Rad52 homologue, Sgs2, the Werner’s syndrome (WRN) helicase homologue, and Tel1, a yeast ATM-like orthologue ([37],[38, 43]). rad51rad50 double mutants effectively eliminate both pathways of suppressors (Chen et al 2001).

Interestingly, a break-induced replication (BIR) pathway of DNA repair between short inverted repeats requires many of the same gene products as Type II recombination ([45]). Remarkably, only 33 bp of homology is required for efficient recombination ([45], [46]). These shared requirements and the low level of homology within telomeric tracts argue for a mechanistic relationship between short sequence recombination and Type II recombination. (Figure 2, [43],[47]).

Telomere Length Homeostasis. In most organisms, telomeric sequences are replicated imprecisely. Even an individual chromosomal end varies in size among different cells of a population ([48],[49],[50]). However, the average telomere size is tightly regulated, since tracts in most organisms are clustered within discrete size distributions ([15],[48],[49],[50]). In yeast, individual telomeres have tract sizes within 50 bp of a genetically determined mean size ([14], [15]). Telomeres are subject to mechanisms of addition that are inversely proportional to telomere size, thereby maintaining an average telomere length. ([31],[51],[52],[53],[54]). Multiple pathways underlie the maintenance of telomere tract size. First, the C-terminus of yeast Rap1 appears to be counted until wild type size is attained possibly due to the formation a thermodynamically stable structure ([51]). In contrast, longer telomeres shorten slowly to the optimal structure due to replicative and nucleolytic loss [52]). Second, in some contexts, Rap1 may act as a more direct negative regulator of telomerase ([55]). This negative regulation may be mediated through a switch or switches to an open chromatin state conducive to telomerase access or recruitment ([55]). The third mechanism, TRD, returns over-elongated telomeres to wild-type size through an intrachromatid recombination process. This process, the focus of this proposal, appears to be maintained in eukaryotes from yeast to humans, where it may act during apoptosis and in pre-oncogenic states ([56], [57]).

An Equilibrium between Telomerase and End Protection
Lundblad and colleagues have proposed a model for the coordination between capping and replication. One variation of this model is depicted in Figure 3 ([31],[58]).

The C-strand of a fully replicated telomere is degraded by an exonuclease in late S phase to give rise to a telomere containing a >30 bp 3’ overhang. Cdc13 binds and recruits Stn1 and Ten1 to the single stranded overhang. Both Stn1p and Cdc13 may be responsible for the binding of Ten1p. The Ku subunits that bind to the duplex telomere sequence may also functionally associate with Cdc13 and with TLC1 ([59]; [5]). The resulting structure would constitute the telomere end-protection complex that represses further nucleolytic degradation. The telomerase holoenzyme is converted to its active form and both Ku70 and Est1 associate with TLC1 to positively regulate telomerase ([5] [60]). Stn1 subsequently competes with Est1 for overlapping sites on Cdc13, displacing Est1 and telomerase. Stn1 reassociates with Cdc13 directly forming a trimeric Cdc13–Stn1–Ten1 that permits second strand synthesis.

Regulating Second Strand Telomere Synthesis
Second-strand synthesis is carefully coordinated with telomerase activity [31]. Biochemical and genetic associations have been observed between Cdc13 and both pol a and Est1 ([61], [62]), suggesting that their recruitment controls leading/lagging strand homeostasis[63], [64]). Furthermore, telomerase activity on short telomere substrates in late S/G2 cells require DNA primase, pol a, and pol d; a clear demonstration of the need for coordinated GT and CA strand synthesis ([65]). Recent data indicate that Stn1 and the polymerase a regulatory subunit Pol12 interact with and play an as yet critical but undefined function in this coordination (D. Shore personal communication).

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