Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Abstract

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSbeta and endonuclease activity of MutLalpha. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.

Figure 1

Experimental system to study chromosomal fragility induced by expanded tracts of GAA/TTC repeats. The breakage at the location of GAA/TTC tracts can lead to 43 kb telomere-proximal deletion resulting in Can^RAde⁻ clones. In a separate set of strains, the lys2-8 allele was integrated into chromosome III, allowing us to measure the level of homologous recombination induced by GAA/TTC repeats. The ‘X' denotes a recombination event generating a wild-type LYS2 allele.

Figure 2

Structural analysis of chromosomal arm loss events stimulated by (GAA/TTC)₂₃₀ tracts. (A) Analysis of rearranged chromosome Vs in Can^RAde⁻ isolates by CHEF gels and Southern blotting. The right arm of chromosome V was highlighted using a MET6-specific probe in Southern analysis. Lanes labelled with ‘wt' are strains containing wild-type chromosome V with (TTC)₂₃₀ and (GAA)₂₃₀ repeats. Lanes T-1 to T-12 are Can^RAde⁻ isolates from (TTC)₂₃₀ strains. Lanes A-1 and A-12 are Can^RAde⁻ isolates from strains with (GAA)₂₃₀ repeats. The primary GCR classes are labelled in red. (B) CGH and breakpoint analysis of the most frequent rearrangements resulting from (TTC/GAA)₂₃₀-mediated breaks. Upper panels are the microarray analysis of arm loss events. DNAs from experimental strain and control strain were labelled with different fluorescent nucleotides and hybridized in competition to DNA microarrays with yeast genes and intergenic regions. Each vertical bar corresponds to one ORF in Watson (upper bars) and in Crick (bottom bars) orientations. Colour coding is as follows: grey, repeated genomic elements; yellow, sequences present in the same dosage in the wild-type and control strains; red, sequences that were duplicated in the experimental strain relative to the control; blue, sequences that were deleted in the experimental strain relative to the control. Only those chromosomes that had a deletion or duplication are shown in this figure. Complete data for these experiments is online at GEO database (accession number GSE11425). Bottom panels depict the structure of the translocation breakpoints on chromosomes I and XI. The donor sites for BIR are shown. Blue and red arrows indicate the breakpoint junctions between GAA/TTC tracts from chromosome V and GAA/TTC-rich regions on donor chromosomes (examples are shown). The left panel is the analysis of a major class of GCRs in (TTC)₂₃₀ strains (isolates T-1, T-3, T-4, T-5, T-8, T-11 and T-12). The right panel is the analysis of a major class of GCRs in (GAA)₂₃₀ strains (isolates A-1, A-2, A-3, A-5, A-6, A-7, A-9, A-10, A-11 and A-12). The complete analysis of the breakpoints for all isolates is presented in the Supplementary Table 1.

Figure 3

2D analysis of replication intermediates in strains containing GAA/TTC repeats. Neutral/neutral 2D electrophoresis was used to resolve unreplicated molecules and Y-like structures. Replication initiated at ARS507 proceeds from right to left through the region containing the repeat tracts. Cleavage with AflII positions the GAA/TTC repeats on the long shoulder of the Y-arc. The 4 kb AflII-digested LYS2 fragment was used as a probe in Southern blot hybridization. Accumulation of the replication intermediates leads to the appearance of bulges on the replication arc. Replication pausing zones are indicated by brackets. Arrows point to the Y-arc interruptions coinciding with the centre of the GAA tracts.

Figure 4

Induction of GCR and the ability to block fork progression by GAA/TTC repeats are affected by their orientation relative to the origin of replication. (A) GCR rates of original and flipped constructs. The schematic diagram of the original and the flipped LYS2 cassette containing GAA/TTC tracts is shown on the left. The corresponding GCR rates are shown on the right. (B) Replication fork progression across flipped GAA/TTC tracts. 2D analysis was performed as described in Figure 3. The replication pause zone across the flipped TTC tracts is indicated by brackets.

Figure 5

Chromosomal fragility at GAA/TTC tracts requires MMR. (A) MMR mutants strongly affect GCRs induced by (GAA)₂₃₀ tracts. Values are median rates determined in fluctuation tests using at least 14 cultures. Error bars indicate 95% confidence intervals. (B) Breakage of chromosome V in strains containing GAA/TTC repeats. The position of the GAA/TTC tracts on chromosome V is shown. Chromosomes were separated on the CHEF gel, transferred to a nylon membrane and hybridized with DSF1-specific probe to highlight the intact chromosome (∼585 kb) and broken fragment (∼43 kb). λ ladder was used as a molecular size standard shown on the right. The positions of the marker bands were determined on the ethidium bromide-stained gel prior to Southern blot hybridization. The lanes are: 1, wild-type strain with (GAA)₂₀; 2, wild-type strain with (TTC)₂₃₀; 3, wild-type strain with (GAA)₂₃₀; 4, Δmsh2 strain with (GAA)₂₃₀; 5, pms1-E707K strain with (GAA)₂₃₀.

Figure 6

Model for chromosomal fragility and rearrangements mediated by triplex-forming GAA/TTC repeats. The GAA/TTC tracts are shown (not to scale) in red. Telomeres (filled rectangles) and centromeres (solid circles) are also shown. Diamonds with arrows are the bidirectional replication forks. A non-homologous chromosome is depicted in blue. GAA/TTC tracts are microsatellites that are prone to slippage during DNA synthesis. In MMR-deficient strains, this instability is manifested as small size repeat variations. We hypothesize that a triplex structure will be adopted preferentially when the GAA repeats are located on the lagging strand template. Triplex can arrest replication progression. We suggest that MMR system recognizes and processes the H-DNA leading to DSBs. DSBs can also be introduced by an alternative, MMR-independent minor pathways indicated by the boxed ‘?'. Following DSB induction, the broken end can be healed through intra-allelic repair (such as NHEJ or single-strand annealing) or homologous recombination with a repetitive tract on the sister chromatid, leading to large size variations. Alternatively, centromere-containing broken fragment can be repaired by BIR with GAA/TTC-rich regions on non-homologous chromosomes, resulting in arm loss and non-reciprocal translocation. Rarely, the broken end can also be capped by de novo telomere addition.