Genome rearrangements caused by interstitial telomeric sequences in yeast

Abstract

Interstitial telomeric sequences (ITSs) are present in many eukaryotic genomes and are linked to genome instabilities and disease in humans. The mechanisms responsible for ITS-mediated genome instability are not understood in molecular detail. Here, we use a model Saccharomyces cerevisiae system to characterize genome instability mediated by yeast telomeric (Ytel) repeats embedded within an intron of a reporter gene inside a yeast chromosome. We observed a very high rate of small insertions and deletions within the repeats. We also found frequent gross chromosome rearrangements, including deletions, duplications, inversions, translocations, and formation of acentric minichromosomes. The inversions are a unique class of chromosome rearrangement involving an interaction between the ITS and the true telomere of the chromosome. Because we previously found that Ytel repeats cause strong replication fork stalling, we suggest that formation of double-stranded DNA breaks within the Ytel sequences might be responsible for these gross chromosome rearrangements.

Keywords: interstitial telomeres; telomere silencing.

Fig. 1.

System used to detect genome instability induced by ITSs. (A) Cassette used to generate strains with ITS sequences. The ∼3-kb cassette contains flanking sequences from chromosome III (black), flanking and coding sequences from URA3 (yellow and red, respectively), intronic sequences from the ACT1 gene (blue), and TRP1 flanking and coding sequences (pale green and dark green, respectively). Telomeric repeats were inserted into the indicated XhoI site within the intron. Small arrows flanking the XhoI site show the positions of primers UIRL1 and UIRL2. Numbers above the cassette indicate the position in the cassette, and numbers below the line are SGD coordinates. (B) Location of the cassette on chromosome III showing various relevant chromosome elements. Large black arrows indicate Ty elements and small arrows show solo delta elements; large purple arrows indicate the telomeres. LAHS, FS1, and FS2 are abbreviations for left-arm hotspot, fragile site 1, and fragile site 2 (17). (C) Representative PCR analysis of 5-FOA^R colonies derived from strain SMY752 that contained the eight-repeat telomeric tract. Primers located within 50 bp of the insertion were used to amplify genomic DNA, and the resulting fragments were separated by gel electrophoresis. Nos. 7, 8, and 9, the amount of Ytel repeats as confirmed by DNA sequencing; np, no PCR product indicative of genomic rearrangements.

Fig. 2.

CHEF gel analysis of chromosome rearrangements in 5-FOA^R isolates of SMY749 that had alterations in the sequences flanking the ITS. (A) Chromosomal DNA from 15 isolates was compared with DNA from the SMY749 control strain (lane C). Lanes labeled “L” and “Sc” contain size standards (Bio-Rad), catenated lambda bacteriophage chromosome and yeast chromosomes from a standard laboratory strain, respectively. The gel was stained with ethidium bromide (Left) and subsequently transferred to nitrocellulose filters and hybridized to LEU2 (Right). The positions of the hybridizing bands in the ethidium bromide-stained gel are boxed, and arrows show the position of chromosome III before the chromosome rearrangement. (B) Locations of the hybridization probes relative to the URA3::Ytel gene. (C) CHEF electrophoresis followed by Southern analysis using the CHA1 hybridization probe. The minichromosome is shown by a double arrow on the right side of the figure.

Fig. 3.

CGH microarray analysis of 5-FOA^R derivatives of SMY749. The results of CGH experiments analyzed with the CGH-Miner program. Genomic DNA isolated from the experimental strain was labeled with Cy5-dUTP, DNA from the control strain without chromosome rearrangements (SMY749) was labeled with Cy3-dUTP, and the samples were mixed and hybridized to microarrays containing all ORFs and intergenic regions. The horizontal line represents the entire length of chromosome III (or II). Green and red show a significant deletion or duplication of sequences, respectively. The approximate SGD coordinates for the labeled deletions and duplications on the indicated chromosomes are Δ1 (III, 76,000–85,000), Dup1 (III, 150,000-right telomere), Dup2 (III, 125,000-right telomere), Dup3 (II, 1–30,000), and Mini (III, 1–76,000). The number of the 5-FOA^R isolate is shown in parentheses. The CGH analysis of the minichromosome was done using a chromosome purified from a CHEF gel.

Fig. 4.

Formation and analysis of class 1 events (terminal inversions). (A) Mechanism for inversion (see text for details). Telomeric repeats are shown as double-stranded DNA molecules, with blue and red indicating the GT-rich and CA-rich strands, respectively. The rectangle labeled “X” indicates the conserved subtelomeric X repeat (1), and the horizontal arrows show the positions of primers used to diagnose the inversion. (B) PCR analysis of class 1 events.

Fig. 5.

Mechanisms responsible for four classes of chromosomal rearrangements (see text for details). (A) Terminal inversion (class 1). (B) Deletion associated with gene conversion (class 2). (C) Deletion/duplication of chromosome III and minichromosome (class 3). (D) Translocation and minichromosome (class 4). ITSs are shown as red triangles, telomeric repeats at chromosome ends are shown as orange triangles, centromeres are shown as circles (white for chromosome III and black for other chromosomes), and Ty and delta elements are shown as black arrows.