Repair of chromosome ends after telomere loss in Saccharomyces

Abstract

Removal of a telomere from yeast chromosome VII in a strain having two copies of this chromosome often results in its loss. Here we show that there are three pathways that can stabilize this broken chromosome: homologous recombination, nonhomologous end joining, and de novo telomere addition. Both in a wild-type and a recombination deficient rad52 strain, most stabilization events were due to homologous recombination, whereas nonhomologous end joining was exceptionally rare. De novo telomere addition was relatively rare, stabilizing <0.1% of broken chromosomes. Telomere addition took place at a very limited number of sites on chromosome VII, most occurring close to a 35-base pair stretch of telomere-like DNA that is normally approximately 50 kb from the left telomere of chromosome VII. In the absence of the Pif1p DNA helicase, telomere addition events were much more frequent and were not concentrated near the 35-base pair tract of telomere-like DNA. We propose that internal tracts of telomere-like sequence recruit telomerase by binding its anchor site and that Pif1p inhibits telomerase by dissociating DNA primer-telomerase RNA interactions. These data also show that telomeric DNA is essential for the stable maintenance of linear chromosomes in yeast.

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.

Figure 2

Southern analysis of stabilized clones. (A) Structure of chromosomes stabilized by telomere addition within URA3. The structure of the left telomere of the test chromosome is shown. The open arrow head represents the ∼350-base pair tract of C_1–3A/TG_1–3 DNA. The solid black rectangle is the 1.1-kb HindIII-SmaI fragment that contains the URA3 open reading frame; the bent arrow denotes the translational start site for URA3. The region between the telomeric tract and the end of the HindIII-SmaI URA3 fragment is a 308-base pair EcoRI-HincII fragment containing the MAT locus with its HO recognition site. There are 50 bps between the HO cut site and the start of the telomeric tract. Sites for relevant restriction enzymes and for the HO endonuclease are indicated. DNA from two stabilized clones (lanes 1 and 2) or the starting disome strain (lane 3) was digested with StuI or NcoI, separated on a 1% agarose gel, and analyzed by Southern blotting with the use of a probe for the entire URA3 gene. Asterisks indicate telomeric restriction fragments. The other bands that hybridize to the probe were from either the ura3-52 locus or the portion of the telomeric URA3 that was proximal to the StuI (or NcoI) site. The sites of telomere addition as deduced from this analysis are indicated on the diagram (labeled 1 and 2, for clones in lanes 1 and 2, respectively). M, molecular weight markers (1-kb ladder). (B) PFGE mapping of stabilized clones. Positions of hybridization probes and the relevant NotI site are shown in Figure 1. Probe 1 (detects both copies of chromosome VII) was used in C and D; probe 2 (detects only the test chromosome) was used in B. Lanes 1–7 in B contain undigested DNA from independent stabilized clones isolated in the rad52 strain. NotI-digested DNA from the same clones is shown in C. Lanes 1 and 7 contain DNA from clones stabilized by de novo telomere addition within URA3; lanes 2, 3, and 4 contain clones stabilized by recombination with the endogenous copy of chromosome VII; lanes 5 and 6 contain DNA from clones stabilized near the (CA)₁₇ tract. Lane 8 contains DNA from the starting disome strain; lane 9 has DNA from a haploid strain containing only the endogenous copy of chromosome VII. Lanes 1–8 in D contain NotI-digested DNA from independent stabilized clones isolated in the rad52 pif1-m2 strain. Each clone was stabilized by telomere addition internal to URA3. Lane 9 contains DNA from the starting disome strain, and lane 10 contains DNA from a haploid strain containing the endogenous chromosome VII.

Figure 3

Positions of de novo telomere addition sites near or within URA3. (A) Positions of independent stabilization events from the rad52 strain are denoted by circles or squares. Circles are clones isolated in the experiment used to determine the frequencies of stabilization reported in Supplementary Table 1A. Squares indicate clones isolated in independent stabilization experiments. Open symbols denote clones whose positions were determined solely by Southern analysis. Closed symbols denote the positions of clones that were mapped precisely by DNA sequencing. The asterisk marks the position of an 11-base pair telomere-like sequence in URA3. The exact sites of telomere addition for these clones are shown in B, which presents the sequence of the strand running 5′ to 3′ from the end toward the center of the chromosome of the URA3 sequence, starting with the SmaI site used to clone the gene. The telomere is distal to the SmaI site. The TAA termination site for Ura3p is in bold. Black arrows indicate the sites of telomere addition in four independent stabilization events isolated in the rad52 strain. Open arrows indicate the sites of telomere addition in 15 independent stabilized clones isolated in the rad52 pif1-m2 strain. In three cases, multiple clones had the same site of telomere addition: in these cases, the number of events at the site is indicated. The 9- and 11-base pair tracts of telomere-like DNA are in bold and underlined. Tracts of telomere-like DNA of 5–7 base pairs are underlined with dotted lines. (C) Positions of independent stabilization events from the rad52 pif1-m2 strain; symbols are the same as in A. The exact sites of telomere addition for these clones are marked by open arrows in B. The positions labeled 1, 2, and 3 are shown in expanded form in D, the sequence of the 100 bps internal to the HO cut site. Telomere addition at these sites yielded Ura⁺ cells. The arrow indicates the points of transition between the sequence of the test chromosome and the added telomeric DNA. The bold, underlined bases fit the consensus for telomeric DNA. (E) Compares the sequence around the HO cut site in the test chromosome with the sequence for this region in three independent Ura⁺ stabilized clones isolated in a rad52 strain. The dashes in the sequences indicate missing bases.

Figure 4

Positions of de novo telomere addition sites internal to URA3. The ∼255-kb NotI fragment from the left end of the test chromosome is depicted in A and B. The open arrowhead represents the telomere. The symbols indicate the sites where new telomeres were added in independent stabilized clones isolated in the rad52 strain (A) or the rad52 pif1-m2 strain (B). Circles are clones isolated in the experiments used to determine the frequencies of stabilization reported in Supplementary Table 1A. Squares indicate clones isolated in independent stabilization experiments. Open symbols denote clones whose positions were determined solely by PFGE and Southern analysis. Closed symbols denote the positions of clones that were mapped precisely by DNA sequencing; the sequence at these sites is presented in C and D. C and D are the sequence of two sites ∼50 kb from the left telomere of chromosome VII. Arrows indicate the sites of telomere addition in five independent stabilized clones isolated in the rad52 strain. The sequence in C is 200 base pairs, starting at base pairs 67,000 from the left telomere of chromosome VII (all sequence coordinates are from the Saccharomyces genome database; http://genome-www.stanford.edu/Saccharomyces). A 31-base pair CA-rich tract that resembles but does not match the consensus sequence for telomeric DNA is underlined. The dotted line denotes a 7-base pair stretch of telomere-like DNA. (D) Shows 150 base pairs, starting at base pairs 69250. The (CA)₁₇ tract is in bold and underlined. There were no other stretches of 6 bps or greater of telomere-like DNA in the 2.4-kb segment of chromosome VII between coordinates 67,000–69,400.

Figure 5

Distribution of telomere-like sequences in yeast. (A) Distribution of telomere-like DNA, sequences that match the C_1–3A/TG_1–3 consensus sequence for yeast telomeric DNA, within the distal ∼220 kb of the test chromosome VII. Only tracts of nine bases or longer that are in the correct orientation to support telomere addition are shown. The most distal 9- and 11-base pair tracts are from URA3 and were only on the test copy of chromosome VII. When URA3 is in its normal position near the centromere of chromosome V, these 9- and 11-base pair tracts are not in the same orientation as telomeric DNA. There was one 9-base pair tract of telomere-like DNA on the endogenous copy of chromosome VII that was eliminated during construction of the test chromosome. (B) Exact sequence and position of 9-base pair or longer tracts of telomere-like DNA in the same (left) or different (right) orientation as telomeric DNA are shown. The coordinates for each tract are based on the sequence of intact chromosome VII, which is ∼15 kb longer than the test chromosome. (C) Presence of tracts of telomere-like DNA of 15 bps or greater in the yeast genome. Chromosomes I (230 kb), III (317 kb), IX (440 kb), X (745 kb), and XI (670 kb) had no telomere-like tracts >14 bps. Only tracts in the same orientation as telomeric DNA are shown. The subset of C_1–3A/TG_1–3 tracts that are (CA)_n DNA are marked by arrows. All tracts >35 base pairs are adjacent to Y′ subtelomeric repeat sequences, with the exception of the (TG)₃₁ tract located around position 210 kb on chromosome VI. ♦, 15–19 base pairs; □, 20–24 base pairs; ▵, 25–29 base pairs; ●, 35–39 base pairs; *, >50 base pairs; ↓, CA tracts.

Figure 6

Model for de novo telomere addition after chromosome breakage. The striped and spotted rectangles represent, respectively, the C and G-strands of a long stretch of telomere-like DNA, such as the (CA)₁₇ tract on chromosome VII. Small closed circles represent the G-strand of very short, 3–9-base pair stretches of telomere-like DNA. After the HO endonuclease excises the telomere, the break is degraded, with degradation of the 5′ end proceeding faster than the 3′ end (denoted by dotted line). These short TG_1–3-tracts can recruit telomerase, either by simple base pairing with telomerase RNA (left) or by first binding Cdc13p (Lin and Zakian, 1996; Nugent et al., 1996), which then recruits telomerase (Evans and Lundblad, 1999; Qi and Zakian, 2000; Zhou et al., 2000a). The TG_1–3/telomerase RNA association is disrupted by the Pif1p helicase, thus inhibiting de novo telomere addition. In the absence of telomerase lengthening, this 3′ single-strand tail is channeled into recombination. The G-rich strand of a long stretch of telomere-like DNA (dotted rectangle) can recruit telomerase by binding the anchor site of telomerase (right). The 3′ end of the DNA break is then brought to the active site of telomerase by looping out of the intervening DNA allowing elongation of the 3′ end by addition of TG_1–3 DNA (spotted rectangle).