Long CTG Tracts from the Myotonic Dystrophy Gene Induce Deletions and Rearrangements during Recombination at the APRT Locus in CHO Cells

Abstract

Expansion of CTG triplet repeats in the 3 untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG) 17 , (CTG) 98 , and (CTG) 183 repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG) 98 and (CTG) 183 , 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG) 98 and (CTG) 183 cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG) 17 repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.

Selectable System for Monitoring the Instability of CTG/CAG Triplet Repeats in Mammalian Cells

“…In some cases, stable GPT ϩ transformants were isolated and screened by Southern blotting for single-copy integrants that carried the adjacent APRT gene, and then the ratio of APRT ϩ to GPT ϩ colonies was determined by plating the cells under appropriate selection conditions (Table 1). In both assays, all lengths of CTG repeat yielded approximately equal numbers of APRT ϩ and GPT ϩ colonies, indicating that repeats in the CTG orientation do not interfere with APRT expression, as expected from our previous results (33). By contrast, the outcome with repeats in the CAG orientation depended on the length of the tract.…”

“…4A). Because a small degree of variability is introduced into the repeat length by propagation of the cell lines and by PCR amplification (33), it is difficult to know the exact 5Ј boundary of the CAG exon. We assume that one CAG repeat provided the consensus AG that defines the 3Ј end of the adjacent intron.…”

“…Because APRT and HPRT genes can be selected in either direction, it should be possible, in principle, to design selection assays for expansion. For example, it may be feasible to use the APRT(CAG) 33 cell line, which is APRT ϩ , to select for expansions to longer repeat tracts, which are APRT Ϫ . Fortuitously, the crossover point in this assay-33 repeats-is at the upper range of normal in the progression of the human disease and is perhaps ideally suited for studying the initial instability that leads to the disease state.…”

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

“…In yeast, CAG repeats ranging from 130 to 250 units were shown to induce DSBs at a high enough frequency to be directly detectable by Southern blot analysis (42). In mammalian cells carrying CAG repeats of 98 or 183 units, breaks were detected indirectly via stimulation of gene rearrangements, albeit with a lower frequency than in yeast (28). A low frequency of spontaneous breakage is also supported by the absence of fragile sites associated with the myotonic dystrophy locus from patient cells that carried several thousand CAG repeats (43).…”

“…In both yeast and mammalian cells, CAG repeats have been shown to be prone to DSBs (27,28). To examine whether DSB repair contributes to CAG repeat instability, we designed 2 ZFNs to cooperatively bind and cleave CAG repeat tracts.…”