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

Robert D. Wells,Laura P.W. Ranum,Marek Napierala,Ruhee Dere

doi:10.1074/jbc.m406415200

Robert D. Wells, Laura P.W. Ranum + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m406415200

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Abstract

The genetic instabilities of (CCTG.CAGG)(n) tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients. DM2 is caused by an expansion of the repeat from the normal allele of 26 to as many as 11,000 repeats. Genetic expansions and deletions were monitored in an African green monkey kidney cell culture system (COS-7 cells) as a function of the length (30, 114, or 200 repeats), orientation, or proximity of the repeat tracts to the origin (SV40) of replication. As found for CTG.CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts. Also, the expansions and deletions predominated when cloned in orientation II (CAGG on the leading strand template) rather than I and when cloned proximal rather than distal to the replication origin. Biochemical studies on synthetic d(CAGG)(26) and d(CCTG)(26) as models of unpaired regions of the replication fork revealed that d(CAGG)(26) has a marked propensity to adopt a defined base paired hairpin structure, whereas the complementary d(CCTG)(26) lacks this capacity. The effect of orientation described above differs from all previous results with three triplet repeat sequences (including CTG.CAG), which are also involved in the etiologies of other hereditary neurological diseases. However, similar to the triplet repeat sequences, the ability of one of the two strands to form a more stable folded structure, in our case the CAGG strand, explains this unorthodox "reversed" behavior.

Highlights

The genetic instabilities of (CCTG1⁄7CAGG)n tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients
As found for CTG1⁄7CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts
Myotonic dystrophy type 2 (DM2)[1] is a dominantly inherited neurological disease caused by the expansion of a (CCTG1⁄7CAGG) tetranucleotide repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene (1)

Summary

EXPERIMENTAL PROCEDURES

Construction of the (CCTG1⁄7CAGG)n Containing Shuttle Vector—The (CCTG1⁄7CAGG) tetranucleotide repeats (1) were obtained from three pCR2.1TOPO derivatives containing 30, 114, or 200 repeats. The (CCTG1⁄7CAGG)n tracts were excised using an EcoRI digest, purified on a 5.5% polyacrylamide gel, and ligated into the MfeI site of the same shuttle vector This fragment had 11 bp of pCR2.1TOPO vector flanking sequence on one side and 9 bp of vector flanking sequence on the other side of the repeating tract. Genetic Instabilities by Individual Colony Analyses—The episomal DNA, obtained after transfection in COS-7 cells cultured for either 48 h or 2 weeks, was transformed into E. coli HB101 and plated on LB plates containing ampicillin (100 ␮g/ml). The genetic instability was measured as a change in the length of the (CCTG1⁄7CAGG)n repeat containing fragments when compared with the size of the insert excised from the parental plasmid and the 1-kbp DNA ladder size standard (Invitrogen). The gels were analyzed using a Amersham Biosciences Storm 820 (Amersham Biosciences) PhosphorImager and quantitative analyses were performed using ImageQuant version 5.1

RESULTS

Number of repeats

DISCUSSION