Generation of telomere-length heterogeneity in Saccharomyces cerevisiae.

Abstract

Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats. We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication. For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone. This type of variation was independent of the major yeast recombination pathway. The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication. Lengths of individual telomeres within a single clone varied independently of each other. Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately. These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication.The DNA of each eukaryotic chromosome consists of a long linear DNA molecule that requires special structures at its ends. The function of these structures, the telomeres, is 2-fold: they must allow the cell to distinguish these stable chromosome ends from unstable chromosome breaks, and they must solve the problem of incomplete replication at the 5' end of each DNA strand caused by the requirement for a templated primer by DNA polymerases. The (5, 9). Based on these findings, we proposed previously that these repeats were added in a nontemplated fashion by an enzyme analogous to terminal transferase, and that an equilibrium between such a lengthening activity and a shortening of telomeres caused by incomplete replication or exonucleolytic degradation was the key feature of telomere structure and function (5). The discovery of such an elongation activity in Tetrahymena, which in vitro adds G4T2 repeats onto synthetic DNA oligomers representing several known telomeric sequences (10), strongly supports this model of telomere replication.In this paper, we report an analysis of the behavior of individual chromosomal telomeres during cell divisions in haploid S. cerevisiae, which directly tests some predictions of the dynamic equilibrium model. Other studies have addressed genetic factors in yeast that affect the average number of telomeric repeats at all of the chromosome ends coordinately (11-13). Here and a strain with a cdc17 mutation derived from it (a adel ade2 ural lys2 gall cdc17-1) were obtained from the Yeast

Telomere length constancy during aging of Saccharomyces cerevisiae

“…During aging, the telomeres were stable and were maintained at a constant length by the balance of activities (29) that lengthen or shorten the telomeres. The lengthening process is due either to recombination (26) or to the activity of telomerase (10,11,30 (15).…”

Tetrahymenamicronuclear sequences that function as telomeres in yeast

“…Selective media were prepared as in ref. 23. Restriction digestion, gel electrophoresis, radcolabelling, and blot hybridization were carried out under standard conditions or as described (22). Hybridizations were stringent (4XSSC at 650C).…”

“…Yeast transformations were performed according to Hinnen et al (20), using as the host S. cerevisiae strain LL20 (21). Preparation of whole cell DNA from yeast transformants and DNA enriched for extrachromosomal plasmids have been described (22,8). Selective media were prepared as in ref.…”

Structure and variability of human chromosome ends.

“…A similar gradual telomere elongation, compatible with the addition of telomeric repeats by telomerase, occurs in continuously growing T. thermophila (25) and a cell cycle mutant (cdc17) of S. cerevisiae (11). In wild-type S. cerevisiae (41), however, and in T. thermophila grown in batch cultures (25), the tandem array of telomeric repeats is maintained at constant length. At least four genes (CDC17, EST], TEL1, and TEL2 [11,26,27]) govern the length and stability of yeast telomeres; their mode of action is not understood.…”