Hypermethylation of telomere-like foldbacks at codon 12 of the human c-Ha-ras gene and the trinucleotide repeat of the FMR-1 gene of fragile X.

Runs of G residues on the G-rich strands of 30mers from the region spanning codon 12 of c-Ha-ras appear to be protected against chemical modification by dimethylsulfate. This suggests that the G-rich strand might spontaneously form a Hoogsteen-paired quadruplex, which is characteristic of telomere-like DNA sequences. In this report we show that the predominant species in 1:1 mixtures of complementary 30mers from this region are duplex DNA and a smaller amount of unimolecular foldback formed by the C-rich strand. Foldbacks of this type resemble structures first observed in the C-rich strand of telomeric DNA and also occur at the CCG triplet repeat present in the FMR-1 gene of human fragile X syndrome. Foldbacks from the C-rich strand of c-Ha-ras and the FMR-1 triplet repeat are exceptional substrates for the human methyltransferase in isolation. Substituting inosine for guanosine alters the secondary structure of the folded oligomers and dramatically reduces their ability to serve as substrates for the human methyltransferase, suggesting that secondary structure is required for recognition by the enzyme. These findings suggest that one mechanism by which methyl groups accumulate in the c-Ha-ras region of chromosome 11 during carcinogenesis and at the FMR-1 locus during repeat expansion at fragile X may be structurally induced de novo methylation at sites undergoing local conformational change. Such methylation might serve to mark unusual structures for repair. In the absence of repair, asymmetrically methylated duplexes produced by resolution of the unusual structures would be rapidly converted to symmetrically methylated duplexes through the methyl-directed activity also carried by the human methyltransferase.