Chromosome healing through terminal deletions generated by de novo telomere additions in Saccharomyces cerevisiae

Abstract

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3' end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a low-complexity guide RNA sequence.

Fig. 1.

Nonrandom distribution of de novo telomere additions. (a) The assay was generated by replacing HXT13 with URA3 in haploid strains, and GCRs were isolated by selection against CAN1 and URA3. Breakpoints must occur within or between CAN1 and the most centromeric essential gene, PCM1.(b) Histogram in which the breakpoints for the 534 de novo telomere additions isolates are displayed along chromosome V as the number of breakpoints present in 50-bp (light gray) and 500-bp (dark gray) windows. (c) Breakpoint sequences can be divided into three parts: sequences that are unambiguously chromosomal, sequences that could be chromosomal or telomere-derived (junction sequences), and sequences that are unambiguously telomeric. The first identifiable telomere nucleotide is the position between 1 and 2, and the last identifiable chromosomal nucleotide breakpoint is the position between 2 and 3. Three percent of telomeres are added to non-GT targets, so there is no junction sequence and the first and last identifiable positions are identical. (d) The 534 de novo telomere addition breakpoints are nonrandom. Cluster size is the number of times a specific site was targeted by de novo telomere addition, and the number of breakpoints found in each cluster size is plotted. When analyzed by last identifiable nucleotide (light gray), the average cluster has 4.9, with a minimum of one breakpoint and a maximum of 18; when analyzed by the first identifiable nucleotide (dark gray), the average is 6.5 and the range is from 1 to 20 breakpoints per nucleotide. A Poisson distribution predicts an average cluster size of 1.02 assuming each nucleotide is an equally likely target.

Fig. 2.

TG bias at the sites of de novo telomere additions. The percentage of G + T (light gray) and A + C (dark gray) nucleotides present at each position relative to the site of de novo telomere addition as defined by the last identifiable nucleotide is graphed for all additions (n = 534) (a), additions at sites used only once (n = 146) (b), and additions at sites used four or more times (n = 243) (c). (d) Histogram of the individual dinucleotide 5′ to last identifiable breakpoint nucleotide method shows a bias toward GG, GT, and TG, but not TT. No such bias exists for histograms generated from the first identifiable breakpoint.

Fig. 3.

Targets sites are short GT-rich sequences. (a) The number of telomere additions in 50-bp windows (black bars above chromosome) between CAN1 and PCM1 compared to the number of four-base sequences made up of two adjacent TG, GT, or GG dinucleotides (e.g., four contiguous bases, gray bars below the chromosome). (b) The number of telomere additions at sequences containing solely G/T (gray) or A/C (black) nucleotides as a function of sequence length. The length includes bases that would be truncated if the breakpoint fell in the middle of the runs of G/T or A/C nucleotides. Despite the preference for G/T-rich stretches, the numbers of different G/T-rich and A/C-rich stretches between CAN1 and PCM1 are roughly identical and are close to distributions that would be predicted by random distribution (data not shown).

Fig. 4.

Addition sequence registration indicates precise TLC1 registration. (a) Twenty-three different 11-nt telomere addition sequences are observed five or more times after the last identifiable chromosome V breakpoint nucleotide and involve 80% (426 of 534) of all breakpoints. The longest homology to the TLC1 RNA template region is underlined. The most common sequence, seen 80 times, is the ADD1 sequence (28). The first nucleotide in TLC1 homology added after the breakpoint is indicated in the start column; for GGTGTGGGT, the sequence can either register with G¹⁴ or G⁹; however, the last junction nucleotide is T, suggesting that G¹⁴ is most likely the correct. (b) Telomere additions from a were grouped by start position. The right circle is the last identifiable breakpoint, which is placed by using the start position relative to the TLC1 homology. The left circle is the average position of the first identifiable breakpoint for all telomeres in the group. The horizontal line is the average TLC1 homology for all telomeres both before and after the last identifiable breakpoint. (c) The sequence families added after ADD1 are illustrated as in b and correspond to six of the families illustrated in b. The first nucleotide found after ADD1 is indicated by the circle, and the solid line represents the average length of homology with TLC1. It was possible to assign 56 of the 80 sequences; of the remaining, two aligned starting at nucleotide -1, one aligned starting at nucleotide -3, and the remaining 21 could not be assigned because insufficient sequence was available.

Fig. 5.

Heterogeneity in de novo telomere additions suggests both one- and two-step mechanisms. Telomere additions with at least three T or G nucleotides before the last identifiable breakpoint were analyzed by the potential position of the first nucleotide after the breakpoint within the TLC1 homology; each histogram is positioned at this first nucleotide. Registration was determined by using only sequence after the last identifiable breakpoint (“?” were not interpretable). Stars indicate addition registrations predicted based on annealing between TLC1 and the end of the junction sequence (sequence between the first and last breakpoint nucleotides; see Fig. 1c). Triangles indicate addition registrations that would be predicted based on annealing TLC1 with the junction sequence ends but are not observed. Surprisingly, many additions with GGG, TGG, and GGT before the last identifiable breakpoint are observed to initiate at positions lacking homology with the junction sequences (histograms without stars); however, these cases can be explained by two annealing-extension-disassociation cycles in which each annealing position is still controlled by three to four bases of homology of the 3′ end with the TLC1 template. A specific example is illustrated in Fig. 6.

Fig. 6.

Proposed mechanism for de novo telomere addition illustrated by addition of ADD1 onto a TGG breakpoint by two cycles of annealing and synthesis. In vitro studies indicate telomerase requires a 3′ single-stranded substrate (16) that could be revealed by resection of the broken chromosome V (44). Initial annealing of TGG at the preferred location allows up to five bases in the first annealing-synthesis-dissociation cycle; synthesis of more than five bases would not generate an ADD1 addition sequence. Dissociation after either ¹⁰G¹⁰ or ¹⁰GT¹¹ are added will generate new ends that will preferentially reanneal to the initial annealing registration (Fig. 5) and therefore give the appearance of high processivity through this region when analyzing only bulk telomeric sequences. On the other hand, dissociation of longer fragments generated by addition of ¹⁰GTG¹², ¹⁰GTGT¹³, ¹⁰GTGTG¹⁴, or ¹⁰GTGTGG¹⁵ will generate new ends that will preferentially allow reannealing to a second, common registration on the TLC1 template (only the specific case of ¹⁰GTGT¹³ addition in the first cycle is illustrated). Synthesis in the second registration is sufficient to add final nucleotides of the 11-nt ADD1 sequence. The robustness of reannealing of potential intermediate sequences to the first and second registrations on the TLC1 template explains the high frequency of ADD1 additions (Fig. 4a).