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. 2003 May;23(9):3152-62.
doi: 10.1128/MCB.23.9.3152-3162.2003.

Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells

James L Meservy  1 , R Geoffrey Sargent, Ravi R Iyer, Fung Chan, Gregory J McKenzie, Robert D Wells, John H Wilson
Affiliations

Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells

James L Meservy et al. Mol Cell Biol. 2003 May.

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.

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Figures

FIG. 1.
FIG. 1.
Structures of the APRT locus in parental CHO cells and in colonies isolated under various selections. (A) Molecular structures of the tandem duplication of APRT sequences at the endogenous locus. The locations of the CTG triplet repeats in the parental cell lines are shown above their common site of insertion in the second intron of the downstream, functional APRT gene (the five exons of APRT are shown as boxes). The sequences immediately surrounding the upstream FRT site (black triangle) and downstream FRT site (open triangle) are different and therefore distinguishable. The upstream copy of APRT is nonfunctional by virtue of a truncated fifth exon and a point mutation in exon 2 that eliminates an EcoRV site (filled box). The upstream and downstream copies share 6.8 kb of homology indicated by brackets: 4.5 kb upstream of the APRT gene (thick line) and 2.3 kb of homology within the gene itself. (B) Molecular structures of the APRT locus in APRT− and TK− APRT− colonies. Products were distinguished by a combination of Southern blotting and PCR analysis. Conversions have a structure like the parental tandem duplication, except that some lose the insert as part of the conversion process (status of the insert is indicated by +/−). CTG+ conversions were distinguished from mutations by PCR analysis. Mutations are assumed to carry point mutations or small deletions elsewhere in the APRT gene; however, they were not further characterized. Crossovers have a single copy of the APRT gene whose digestion pattern depends on whether the insert (+) was retained or lost (−). Rearrangements yield a Southern blot pattern that does not correspond to conversions, crossovers, or mutations.
FIG. 2.
FIG. 2.
Agarose gel electrophoresis of PCR fragments generated by amplification across the CTG repeats in individual colonies isolated from a population of proliferating GS3506 cells. The individual colonies correspond to those in Table 1, and the PCR products shown here (marked by CTG) were the ones that were isolated and subjected to DNA sequence analysis. The bands in the outside lanes show standard 2.5- and 3.0-kb markers from a commercial ladder. To accentuate the slight differences between bands, the fragments were electrophoresed through about 20 cm of gel. The source of the faint band at around 2.5 kb in all lanes is unknown.
FIG. 3.
FIG. 3.
Southern analysis of APRT− colonies isolated from the (CTG)98 cell line, GS3504. (A) Restriction map for conversions and crossovers. Arrows indicate the sites at which BamHI cleaves, and the sizes of the resulting fragments are indicated in kilobases. Because a BamHI site is located adjacent to the CTG sequence in the insert, the Southern blot pattern depends on whether the insert is retained or lost. A conversion that has lost the insert yields fragments of 12.3 and 4.0 kb. If the insert is retained, the 4.0-kb fragment is replaced by a pair of fragments at 1.3 and 3.1 kb. Similarly, a crossover that has lost the insert yields a single band at 4.0 kb, whereas a crossover that has retained the repeat yields bands at 1.3 and 3.1 kb. (B) Southern blot of BamHI-digested DNA from APRT− colonies isolated from the (CTG)98 cell line, GS3504. DNAs from individual colonies were digested with BamHI, and the fragments were resolved by gel electrophoresis and made visible by Southern blotting. Numbers at the side indicate the lengths of fragments in kilobases. Numbers at the top identify the individual colonies. The structures of GS3504-200 and GS3504-203, which are rearrangements, are shown in more detail in Fig. 4.
FIG. 4.
FIG. 4.
Molecular structures of rearrangements. The structure of the APRT locus in the parental (CTG)98 and (CTG)183 cell lines, GS3504 and GS3506, respectively, is indicated at the top. B and H indicate sites of BamHI and HindIII cleavage, respectively. The CTG tracts in these cell lines carry a BamHI site to the left of the CTG sequence and a HindIII site to the right. Numbers to the right identify individual colonies. Southern blotting was carried out on BamHI-digested DNA and on HindIII-digested DNA for each colony. PCR analyses were used to refine estimates of the positions of the ends of the rearrangements. Boxes above the site of the original CTG sequence indicate various inserted sequences. Open boxes in GS3504-658 and GS3506-859 indicate insertions that have not been further characterized. For GS3504-200, GS3504-639, GS3504-657, GS3506-289, and GS3506-295 the rearrangement junction fragment was isolated and sequenced.
FIG. 5.
FIG. 5.
Speculative model for interaction of CTG repeats with homologous recombination. Although the upstream and downstream copies of the two APRT genes are not shown explicitly, the CTG repeats are present in the top strand of the downstream copy, as illustrated. To generate APRT− crossovers by SSA requires a double-strand break between the mutant and wild-type copies of exon 2, that is, to the left of the CTG repeat, as shown. Conversions by SDSA can be generated in a variety of ways that depend on invasion of a 3′ end, priming of DNA synthesis, release of the extended strand, and pairing with sequences on the other side of the break. The depicted events depart from the standard models for SSA and SDSA (52) at the formation of a hairpin by the triplet repeats and cleavage of the hairpin by the Ercc1/Xpf (E/X) endonuclease. It is hypothesized that a single strand ending in a hairpin is compromised for the strand invasion step in the SDSA pathway. If the initial break is in APRT sequences to the right of the repeat, the right-hand end will not have a hairpin and conversion may proceed normally.
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