The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure.
The fragile X mental retardation syndrome is associated with the expansion of trinucleotide 5'-d(CGG)-3' repeats within the FMR1 gene and with hypermethylation of the cytosine residues of these repeats. The expansion and hypermethylation may account for the suppression of the transcription of the FMR1 gene and for the delay of its replication during the cell cycle. Here we show that d(CGG)n oligomers can form a stable Hoogsteen-bonded structure that exhibits properties consistent with those of tetraplex DNA. Oligomers, d(mCGG)n, (n = 4, 5, or 7), at pH 8.0 and in the presence of an alkali metal ion form stable species exhibiting a reduced electrophoretic mobility in nondenaturing polyacrylamide gels. These species are denatured by heating at 90 degrees C for 10 min. With a short d(mCGG)5 oligomer, the slowly migrating species is formed only when the cytosine residue is 5-methylated, whereas with the longer d(CGG)7 it is generated whether or not cytosine is 5-methylated. By contrast, complementary cytosine-rich oligomers do not form analogous complexes. The second-order association kinetics of the formation of the slowly migrating species of d(mCGG)5 suggests that it is an interstrand complex. Formation of intermediate-size complexes between d(mCGG)5 and d(mCGG)7 indicates that the stoichiometry of the slowly migrating structures is tetramolecular. Protection of the complex from methylation by dimethyl sulfate indicates the involvement of the N-7 positions of the guanine residues in Hoogsteen hydrogen bonding, a characteristic of quadruplex structures. If formed in vivo along the expanded and hypermethylated d(mCGG)n stretch, this tetraplex structure could suppress transcription and replication of the FMR1 gene in the fragile X syndrome cells.
Fry
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Unit of Biochemistry, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Loeb
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D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D007477:Ions; D007700:Kinetics; D008297:Male; D008745:Methylation; D009419:Nerve Tissue Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D016601:RNA-Binding Proteins; D012091:Repetitive Sequences, Nucleic Acid
D051860:Fragile X Messenger Ribonucleoprotein 1; D007477:Ions; D009419:Nerve Tissue Proteins; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human