Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Abstract

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC).-(GCC)., are correlated with the ability of the individual G-and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the Gand C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C-C paired.The presence ofa C C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.Simple tandemly repeated DNA sequences are interspersed in both transcribed and nontranscribed regions of chromosomes (1-3). The hypothesis (4) that the unusual DNA structures adopted by these repeats principally determine their specific functions is gaining strength. We have previously described the unusual hairpin structures (5, 6) adopted by a variety of repetitive DNA sequences. Here, we show by NMR and gel electrophoresis that the individual strands from the fragile X triplet repeats, (GGC)n-(GCC)n, form intramolecular hairpins under physiological conditions. In these hairpins, the number of Watson-Crick G-C pairs is maximized in the stem through the formation of G-G or C C mispairs flanked by G-C pairs (Fig. 1). As shown below, these hairpins provide structural basis for three major phenomena associated with the fragile X syndrome (3, 4): (i) the site-specific fragility, (ii) the amplification of the repeat (especially the preferential expansion of the C-rich strand), and (iii) the hypermethylation of the CpG island adjacent to the fragile X gene, FMRI. MATERIALS AND METHODSGel Electrophoresis. Oligonucleotides were fully denatured by heating at 95°C for 2 min in 5 mM Tris/1 mM EDTA buffer, pH 7.5, containing 5 mM or 200 mM NaCl, followed by incubation at 55°C for 10 min and gradual cooling to roomThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Note that in the hairpins of the G-and C-rich strands, the central mismatched G-G or C-C pair in the stem is surrounded by twoWatson-Crick G-C pairs. IITo whom reprint requests should be addressed. 5199

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

“…As mentioned earlier, trinucleotide repeats, like inverted repeats, have been shown to form hairpin structures in vitro, and in vivo results are consistent with the in vitro studies (15)(16)(17)(18)(19). Hairpin structures are substrates for several structure-specific nucleases, the action of which may lead to DSB formation in DNA.…”

“…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)(16)(17)(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).…”

“…Previous in vitro and in vivo studies have demonstrated that single-stranded DNA containing disease-associated trinucleotide repeats can form hairpin structures (15)(16)(17)(18)(19). Hairpin and cruciform or double-hairpin structures are substrates for several structure-specific nucleases in vivo.…”

DNA Secondary Structures and the Evolution of Hypervariable Tandem Arrays

“…These include the 5Ј-CAG-3Ј repeat that is unstable in triplet expansion diseases such as Huntington's disease and myotonic dystrophy (26,28,29,31) and the centromeric satellite sequence (27). Other simple satellites such as the A ϩ T-rich hypervariable sequence in the 3Ј region of the human apolipoprotein B gene (66) also have the potential to form cruciforms and hairpins.…”

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

“…Stretches of (CAG) n and (CGG) n are more prone to secondary structure formation than (AGG) n , (AGT) n , and (CAA) n (3). NMR studies show that both CAG or CTG (4) and CGG (5) form stable hairpin structures, comprising a repeat unit of two GC pairs and a mismatched pair under physiological conditions. However, CAA/GTT repeats form no hairpins in vitro (6,7).…”