Expansion of Interstitial Telomeric Sequences in Yeast

Abstract

Telomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 × 10(-4) per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (ALT).

Fig. 1. System used for the detection of Ytel repeat expansions

A. Cassette used to generate strains with internal Ytel repeats and its location relative to the proximal replication origin (ARS306). A ~3 kb-long cassette was constructed as described in (Aksenova et al., 2013). The cassette contains flanking sequences from chromosome III (black), flanking and coding sequences from URA3 (yellow and red, respectively), intron sequences from the ACT1 gene (blue), and TRP1 flanking and coding sequences (pale green and dark green, respectively). Telomeric repeats (8 or 15 copies) were inserted into the indicated XhoI site (blunted) within the intron of the URA3-Intron gene. Numbers above the cassette indicate the position in the cassette, and numbers below the line are SGD coordinates for S288C reference genome. B. Phenotypes of strains carrying Ytel repeats in the URA3-Intron reporter gene. Suspensions containing approximately equal amounts of cells were plated as drops on three different types of medium: 5-FOA-containing synthetic media, synthetic media without Uracil and complete YPD media. C. Expression of the URA3-Intron gene in strains with insertions of telomeric repeats. We examined expression of the URA3-Intron gene by RT-PCR in SMY751 (eight copies of the telomere repeat) and SMY750 (fifteen copies of telomere repeat). The control strain SMY803 has no repeats in the URA3-Intron gene. The rows labeled URA3 mRNA and URA3 pre-mRNA show the relative amounts of spliced mRNA and URA3 pre-mRNA in these strains. The row marked URA3 3’-RNA shows the total level of the URA3 transcript. The row labeled Actin indicates the RT-PCR products for the control actin mRNA.

Fig. 2. Expansions of the (CCCACACA)₈ repeat

A. PCR analysis of 5-FOA^R colonies derived from strain SMY751. Primers (1819F/1819R) flanking the repeat (Fig. S3) were used to amplify genomic DNA. Numbers above the lines indicate the exact number of units within the repetitive tract as determined by DNA sequencing. “Quick-load” 50 bp DNA ladders (NEB) are shown. B. Distribution of different expansion events observed in strain SMY751 carrying (CCCACACA)₈ repeat. C. Representative PCR analysis of Ura+ colonies derived from strain SMY751. Primers (1819F/1819R) flanking the repeat (see Fig. S3) were used to amplify genomic DNA. Numbers above the lines indicate the exact number of units within the repetitive tract as determined by DNA sequencing. Ladders are the same as in A. D. Distribution of contractions events observed in strain SMY751. E. Rates of expansion and contraction in strain SMY751. Expansions were scored on 5-FOA media, while contractions were scored on Ura-media.

Fig. 3. Genetic control of Ytel repeat expansions

Effect of different knockouts on the expansion of the (CCCACACA)₈ repeat. Rates of expansion and 95% confidence intervals (error bars) were calculated as described in Materials and Methods. Distribution of expanded clones in 12-to-36 independent cultures was studied for each strain. Length of the repetitive tract for each analyzed clone was validated by PCR. Numbers below the confidence intervals reflect fold decrease over the wild type, and numbers above the confidence interval reflect fold increase over the wild type. Red dashed contours designate the range of 10-fold difference with the wild type.

Fig. 4. Mechanisms responsible for Ytel repeat instability in the C-orientation

The replication fork progresses slowly through the repeat bound to Rap1 and other proteins. Tof1 and Csm3 components of the replication pausing complex sense the protein bulge at the repetitive tract and slow replication progression through this region (black pause symbol). The replication slow down can lead to repeat expansions via the PRR pathway or HR pathway.