Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres.

Telomeres are complexes composed of proteins and repetitive, typically GT-rich, DNA sequences that cap the ends of linear chromosomes, protecting them from fusion and nucleolytic attack. Defects in telomere maintenance and function have been implicated in human disease and aging. The telomeric tract contains both double-stranded and single-stranded components, the lengths of which are regulated by a number of proteins. In yeast, the tracts of duplex telomeric repeats are typically a few hundred base pairs in length, with a region of single-stranded DNA at the telomeric tip that is seen in a cell-cycle-specific manner (65). It is at the single-stranded telomeric tip that the addition or loss of new telomeric repeats occurs. Telomeric DNA is normally maintained by telomerase, a ribonucleoprotein containing the template for new telomeric repeat synthesis in its RNA component (reviewed in references 3 and 24). The length of the duplex telomeric tract is regulated via the effects of several proteins (reviewed in references 14 and 51), the most important of which appears to be Rap1p, an essential gene product that is also involved in transcriptional regulation (reviewed in references 42 and 47). Rap1p binds directly to double-stranded telomeric repeats in both the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis (26, 31), where it recruits other proteins such as Rif1p and Rif2p (23, 66). The number of Rap1p molecules bound to the telomere also appear to be “counted,” such that new telomeric repeats are not added when the appropriate number of Rap1p molecules are bound at the telomere (34). The tethering of additional Rap1p C termini to the telomere leads to the shortening of the telomeric repeat tract, indicating that the number of Rap1p molecules is counted as part of the mechanism that regulates telomere length. Rap1p is also known to be able to bend DNA, which may be important to its telomere function (18, 43, 63). Cdc13p, on the other hand, is a single-strand telomeric repeat binding protein that binds telomeres with high affinity and interacts with other proteins that act on telomeric ends (6, 13, 30, 45, 46). Cdc13p positively regulates telomere length through interactions with Est1p and the telomerase complex. It also serves a negative regulatory role through interactions with Stn1p. Est1p may function as a cell cycle regulator of telomerase activity (56). Cdc13p, Stn1p, and Ten1p are essential in S. cerevisiae, where they play crucial roles in forming a protein complex that provides a protective capping function at telomeres (19, 20). Although telomeric repeats in most organisms are 5 to 8 nucleotides long, some yeast species have telomeric repeats that are much longer (10, 37). In K. lactis, the telomeric DNA is composed of perfect copies of a 25-nucleotide repeat. The long, homogeneous repeats make this species ideal for genetic dissection of the functions occurring at the telomere. In order to synthesize the 25-nucleotide K. lactis repeat, a 30-nucleotide template region of the Ter1 RNA is copied. The first 5 nucleotides (terminal repeat positions 1 to 5) are identical to the last 5 nucleotides (terminal repeat positions 26 to 30), which provides telomerase with the ability to base pair with the telomeric DNA for new telomeric repeat synthesis (16). A mutation made in the telomerase template will typically be copied into the telomeric repeat during DNA synthesis (38, 48, 52, 68). It is thus possible to examine the effects of mutating specific nucleotides of the telomeric repeat. This type of analysis is complicated by the fact that both the telomerase template RNA and the telomeric DNA (and thus potentially the complex of telomere-associated proteins) are altered; determining which is the cause of any defects seen can be difficult. However, experiments with S. cerevisiae (49), as well as with K. lactis (38, 40), have shown that altering the telomeric sequence can have dramatic consequences for the cell in terms of telomere length regulation. In K. lactis, point mutations in the TER1 template that produce telomeric repeats with the corresponding change have been shown to cause several different telomere length phenotypes. However, the extent of the domains producing these phenotypes has not yet been determined. Mutations in part of the Rap1p binding site cause telomere elongation and in some instances telomere fusions. Other mutations lead to telomere shortening and elevated levels of subtelomeric recombination (39). Two classes of mutations, to our knowledge, have been reported only for K. lactis: delayed telomere elongation (38), in which telomeres in the mutant cells initially shorten but eventually become very long, and phenotypically silent mutations (41, 50). Both the immediate and delayed telomere elongation phenotypes were recessive, indicating that the elongation is due not to an overactive telomerase but rather to telomeric defects. In this study, point mutations made at each position of the K. lactis TER1 template were used to determine the extents of various telomeric domains. Telomerase template mutations leading to long, uncapped telomeres, were examined in more detail. Some of these mutants are known to have telomeres that are defective at binding to Rap1p, while others are outside the Rap1p binding site. The stability of these long telomeres and their response to high copy number of RAP1 were investigated.