THE FRAGILE-X SYNDROME D(CGG)(N) NUCLEOTIDE REPEATS FORM A STABLE TETRAHELICAL STRUCTURE

The fragile X mental retardation syndrome is associated with the expan sion of trinucleotide 5'-d(CGG)-3' repeats within the FMR1 gene and wi th hypermethylation of the cytosine residues of these repeats. The exp ansion and hypermethylation may account for the suppression of the tra nscription of the FMR1 gene and for the delay of its replication durin g the cell cycle. Here we show that d(CGG)(n) oligomers can form a sta ble Hoogsteen-bonded structure that exhibits properties consistent wit h those of tetraplex DNA. Oligomers, d((m)CGG)(n), (n = 4, 5, or 7), a t pH 8.0 and in the presence of an alkali metal ion form stable specie s exhibiting a reduced electrophoretic mobility in nondenaturing polya crylamide gels. These species are denatured by heating at 90 degrees C for 10 min. With a short d((m)CGG)(5) oligomer, the slowly migrating species is formed only when the cytosine residue is 5-methylated, wher eas 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 o f the formation of the slowly migrating species of d((m)CGG)(5) sugges ts that it is an interstrand complex. Formation of intermediate-size c omplexes between d((m)CGG)(5) and d((m)CGG)(7) indicates that the stoi chiometry of the slowly migrating structures is tetramolecular. Protec tion 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 forme d in vivo along the expanded and hypermethylated d((m)CGG)(n) stretch, this tetraplex structure could suppress transcription and replication of the FMR1 gene in the fragile X syndrome cells.