DNA Secondary Structures and the Evolution of Hypervariable Tandem Arrays

106

Expansion of a d(CGG)nFragile X syndrome is an inherited, X-linked dominant mental retardation disorder affecting about one male in 1500 and one female in 2500 (Webb, 1989;Richards and Sutherland, 1992). This syndrome is frequently associated with a folatesensitive fragile site, Xq27.3, on chromosome X of cells of affected males (Sutherland, 1977). The fragile X syndrome is characterized by a substantial expansion of a d(CGG) trinucleotide repeat located in the 5Ј-untranslated region of a housekeeping gene, FMR1, which was identified at a locus coincident with the Xq27.3 breakpoint (Verkek et al., 1991). Whereas normal individuals have 2-50 copies of the d(CGG) sequence, the trinucleotide is amplified in affected subjects to Ͼ200 -2000 copies Kremer et al., 1991;Oberlé et al., 1991; Nakhahori et al., 1991; Verkek et al., 1991;Yu et al., 1991). Expansion of the d(CGG) repeat is accompanied by methylation of the FMR1 promoter and of the amplified trinucleotide tract (Bell et al., 1991;Vincent et al., 1991;Pieretti et al., 1991;Luo et al., 1993). Subsequent to d(CGG) expansion and hypermethylation, the FMR1 gene becomes transcriptionally silent Hansen et al., 1992;Sutcliffe et al., 1992), and the replication of a chromosomal segment spanning Ն150 kb 5Ј and Ն34 kb 3Ј from the d(CGG) n stretch is delayed (Hansen et al., 1993).The molecular mechanisms that govern d(CGG) n amplification and hypermethylation and that link d(CGG) expansion to the suppressed transcription of FMR1 and to its delayed replication are not known. In addressing these problems, we showed recently that model d(CGG) n oligomers form under physiological conditions a tetramolecular four-stranded structure in a time-dependent and DNA concentration-dependent kinetics. We also found that the multimolecular tetraplex structure of short d(CGG) n tracts is stabilized by 5-methylation of the cytosine residues (Fry and Loeb, 1994). The facile formation of tetramolecular complexes by d(CGG) n raised the hypothesis that single stranded stretches of this tract may also generate unimolecular hairpin or tetraplex structures (Fry and Loeb, 1994). In this communication we use gel mobility analysis and chemical probing to demonstrate that d(CGG) n oligodeoxynucleotides can fold back at a physiological range of salt concentrations, temperatures, and pH values to form hairpin structures. In contrast to the second-order kinetics of formation of tetramolecular quadruplex d(CGG) n (Fry and Loeb, 1994), hairpin structures of this sequence are formed at a zero-order kinetics. Our evidence for hairpin formation by d(CGG) n is sustained by reports by Gacy et al. (1995) and Chen et al. (1995), which were published as this work was completed and that provide NMR evidence to also show the formation of hairpin structures by d(CGG) n and by other trinucleotide tracts.The folding of exposed expanded single strand runs of d(CGG) n during the replication of fragile X cell DNA could entail slippage and trinucleotide expansion. Furthermore, guanine-rich tracts of RNA or DNA that fol...

Section: Discussionmentioning

confidence: 99%

Section: Expansion Of a D(cgg)mentioning

confidence: 99%

The Fragile X Syndrome Single Strand d(CGG)n Nucleotide Repeats Readily Fold Back to Form Unimolecular Hairpin Structures

Nadel¹,

Weisman-Shomer²,

Fry³

1995

106

“…These oligodeoxyribonucleotides were then used together with a primer that contained an EcoRI site and 15 bases complementary to the 3Ј-end of the supF gene to generate a PCR fragment as described previously (9,14). For PCR fragments containing inosine or 7-deazaadenine, dITP or 7-deaza-dATP was substituted for the appropriate dNTP in the PCR.…”

Section: Template Preparation-oligodeoxyribonucleotides Containing Anmentioning

confidence: 99%

“…For PCR fragments containing inosine or 7-deazaadenine, dITP or 7-deaza-dATP was substituted for the appropriate dNTP in the PCR. For the construction of plasmid templates, the PCR product was digested with EcoRI, purified by gel electrophoresis, and then cloned into the plasmid pMS189⌬ as described previously (9,14). Plasmids were propagated in E. coli MBM7070, isolated by alkaline lysis, and purified by CsCl * The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Template Preparation-oligodeoxyribonucleotides Containing Anmentioning

confidence: 99%

The Mouse Ms6-hm Hypervariable Microsatellite Forms a Hairpin and Two Unusual Tetraplexes

Weitzmann

Woodford

Usdin

1998

Self Cite

The mouse Ms6-hm microsatellite consists of a tandem array of the pentamer d(CAGGG) n . This microsatellite is extremely hypervariable, showing a germ line mutation rate of 2.5%/gamete. The mechanism responsible for this instability is not known. The ability to form intrastrand structures is a conserved feature of many hypervariable sequences, and it has been suggested that the formation of such structures might account for instability by affecting DNA replication, repair, or recombination. Here we show that this microsatellite is able to form intrastrand structures as well. Under physiological conditions, the Ms6-hm microsatellite forms a hairpin as well as two different unusual intrastrand tetraplexes. The hairpin forms in the absence of monovalent cation and contains G⅐A, G⅐C, and G⅐G base pairs in a 1:1:1 ratio. In the presence of K ؉ , a tetraplex is formed in which the adenines are unpaired and extrahelical, and the cytosines are involved in C⅐C pairs. In Na ؉ , a tetraplex forms that contains C⅐C ؉ pairs, with the adenines being intrahelical and hydrogen-bonded to guanines. Tetraplex formation in the presence of Na ؉ requires both cytosines and adenines and might reflect the altered internal dimensions of this tetraplex, perhaps resulting from the ability of the C⅐C ؉ pairs to become intercalated in this sequence context. Our demonstration of the stabilization of tetraplexes by hydrogen bonding between adenines and guanines expands the hydrogen-bonding possibilities for tetraplexes and suggests that the category of sequences with tetraplex-forming potential may be larger than previously appreciated.

“…We have recently described the presence of a strong K ϩ -dependent DNA synthesis arrest site in a G-rich region of the chicken ␤-globin gene promoter (18). The sequence of the arrest site is shown in boldface type in Fig.…”

mentioning

confidence: 99%

The Chicken β-Globin Gene Promoter Forms a Novel “Cinched” Tetrahelical Structure

Howell¹,

Woodford

Weitzmann

et al. 1996

We have previously shown that the G-rich sequence G 16 CG(GGT) 2 GG in the promoter region of the chicken ␤-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029 -27035). The K ؉ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K ؉ -dependent intrastrand "cinched" tetraplex. This structure has several unique features including the incorporation of bases other than guanine into the stem of the tetraplex, interaction between loop bases and bases in the flanking region, and base pairing between bases 3 and 5 of the tetrahelix-forming region to form a molecular "cinch." This finding extends the range of sequences capable of tetraplex formation as well as our appreciation of the conformational complexity of the chicken ␤-globin promoter.