Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast

Abstract

Expansion of trinucleotide repeats is associated with a growing number of human diseases. The mechanism and timing of expansion of the repeat tract are poorly understood. In humans, trinucleotide repeats show extreme meiotic instability, and expansion of the repeat tract has been suggested to occur in the germ-line mitotic divisions or postmeiotically during early divisions of the embryo. Studies in model organisms have indicated that polymerase slippage plays a major role in the repeat tract instability and meiotic instability is severalfold higher than the mitotic instability. We show here that meiotic instability of the CAG͞CTG repeat tract in yeast is associated with double-strand break (DSB) formation within the repeated sequences, and that the DSB formation is dependent on the meiotic recombination machinery. The DSB repair results in both expansions and contractions of the CAG repeat tract. E xpansion of trinucleotide repeats has been shown to be associated with a growing number of human diseases (1). Several of these genetic diseases are associated with alterations of the CAG͞CTG (hereafter CAG) repeat tract length. The expansion mutation is highly dependent on the repeat tract length: the longer the repeat tract, the greater the possibility for expansion (2-4). Such mutational changes are dynamic, since the mutated region can undergo further changes in subsequent generations and during the lifespan of an individual.Two mechanisms have been used to explain the expansion of trinucleotide repeats: unequal genetic recombination and error in DNA replication caused by polymerase slippage (5, 6). In the recombination model, unequal crossing-over or gene conversion between triplet repeats either on sister chromatids or on homologs results in an altered tract length. In the slippage model, the replicating strand becomes dissociated, and then it misaligns during reassociation. This misalignment causes the formation of an unpaired sequence either on the template strand or on the nascent strand. A failure to repair the unpaired sequence will result in a DNA molecule containing a larger or smaller number of repeats after the next round of DNA replication. Several studies using model organisms indicate that replication slippage plays a major role in the repeat tract instability (7-14). The latter model is also supported by the in vitro observations that CAG repeats can form hairpin structures, with the CTG hairpin being more stable than the CAG hairpin (15-18). Recent studies with diploid yeast strains containing heterozygous trinucleotide repeat-insertion mutations suggest that trinucleotide repeats in single-stranded DNA are likely to form hairpin structures in vivo (19). During DNA replication, the hairpin formation on the template strand or on the newly synthesized strand would produce a deletion or an expansion of the repeat tract, respectively. This hypothesis is consistent with the observation that CAG-repeat instability depends on the direction in which the replication fork proceeds through the repeat tract (...

Fidelity of Primate Cell Repair of a Double-strand Break within a (CTG)·(CAG) Tract

“…Furthermore, the presence of slipped structures within the repeat-containing end enhanced the preferential deletion of repeats, providing the first line of evidence that processing of slipped structures can lead to repeat instability. Our results are consistent with studies in bacterial (21) and yeast (22)(23)(24)(25) models that propose a role for recombination and DSB repair in trinucleotide repeat instability.…”

“…1 The abbreviations used are: DM1, myotonic dystrophy type 1; DSB, double-strand break; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; nt, nucleotides; STRIP assay, Stability of Trinucleotide Repeats by Individual Product analysis. let repeat tracts are "fragile," such that they are susceptible to DSBs, possibly through endonucleolytic cleavage of secondary structures (22,23). The formation of DSBs within or proximal to the repeats is length-dependent.…”

“…The formation of DSBs within or proximal to the repeats is length-dependent. It has been suggested that subsequent repair of the break can lead to instability of the repeat tract, in some cases yielding large deletions of the repeat and flanking sequences (22)(23)(24). The use of a donor sequence containing (CAG) 98 to repair a DSB lead to integration and expansion of the triplet repeat tract (25).…”

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

“…This model is supported by the observation of double-strand breaks in CTG⅐CAG repeats in yeast (82,83).…”

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…Furthermore, these structures are also recognized by mismatch repair (MMR) (29, 44 -48) and nucleotide excision repair (NER) (49,50); both pathways have been implicated in the stability of the secondary structures, thus influencing the expansion and deletion processes. Also, double strand breaks caused by replication fork arrest or repair of the non-B DNA structures induces repair-mediated recombination that may participate in the expansions observed in both prokaryotic as well as eukaryotic model systems (21,30,(51)(52)(53)(54)(55)(56). Triplet repeat sequences are hotspots for recombination, which may account for the massive expansions found in certain diseases (24 -28, 57, 58).…”