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

Abstract

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...

Human Werner Syndrome DNA Helicase Unwinds Tetrahelical Structures of the Fragile X Syndrome Repeat Sequence d(CGG)

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