Figure 1.

Experimental assay and consequences of telomere loss. Experiments were in otherwise haploid strains that were disomic for chromosome VII. The two copies of chromosome VII, the endogenous and test copies, were constructed as described in Sandell and Zakian (1993). In addition to the markers indicated on chromosome VII, the disomic strain was ade2, ura3-52 and had no HO site at MAT. Immediately next to the left telomere of the test chromosome was the HO recognition site followed by URA3. This insertion was made within the ADH4 gene, the most distal gene on the left arm of chromosome VII, in such a way that ∼16 kb of DNA was deleted. Thus, the test chromosome is ∼15 kb shorter than the endogenous copy of chromosome VII. The HO endonuclease gene was inserted within the ADE3 gene on the endogenous copy of chromosome VII, thereby deleting a portion of ADE3. Hybridization with a probe for the deleted region (probe 2) detects only the test chromosome (Figure 2B). Hybridization with probe 1 detects both copies of chromosome VII (Figure 2, C and D). When cells are grown in galactose medium, the HO endonuclease is expressed and the left telomere on the test chromosome is excised in most cells (Sandell and Zakian, 1993). After telomere loss, the test chromosome was either lost (A), repaired by homologous recombination with the endogenous copy of chromosome VII (B), acquired a new telomere at a site that had previously been internal on the test chromosome (C), or religated in a way that eliminated the HO recognition site (D). The phenotype of cells produced by each of these events is indicated. Although most de novo telomere addition events yielded Ura⁻ cells, in the rad52 pif1 strain, some of these events generated Ura⁺ cells. Theoretically, nonhomologous end joining could yield Ura⁻ cells but no such events were recovered. To be detected in this study, recombination and de novo telomere addition had to occur in the ∼220-kb region distal to LYS5 because stabilized clones were selected on medium lacking lysine. There are four NotI sites on chromosome VII; only the one relevant for the analysis in this article is indicated. Most de novo telomere addition events that occur in a rad52 strain were near a (CA)₁₇ tract indicated on the figure. The figure is not to scale.