FRAGILE-X DNA TRIPLET REPEATS, (GCC)(N), FORM HAIRPINS WITH SINGLE HYDROGEN-BONDED CYTOSINE-CENTER-DOT-CYTOSINE MISPAIRS AT THE CPG SITES -ISOTOPE-EDITED NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY ON (GCC)(N) WITH SELECTIVE (15)N4-LABELED CYTOSINE BASES

Here, we provide a direct proof that the formation of hairpins by (GCC )(n) at the 5'-UTR of the FMR-1 gene offers a mechanism for CpG hyperm ethylation associated with the fragile X syndrome. For this, we have p erformed hetero-nuclear (N-15-H-1) magnetic resonance spectroscopy to probe the structure of the CpG sites in the (GCC)(n) hairpins that are N-15-labeled at the amino (N4) groups of specific cytosine bases. Ana lyses of chemical shift, pH-induced chemical exchange, and NOE pattern of the (15N-labeled) amino protons of cytosine bases reveal that the cytosine bases at the CpG sites are intrahelical and well-stacked with the neighboring G.C base-pairs in the stem. of these hairpins and pro bably form single hydrogen-bonded C.C mispairs. Measurements of pH-dep endent H-1 line-width also demonstrate that the C.C mispairs are more susceptible to open-closure than the G.C base-pairs. Thus, the Cs at t he CpG sites of the (GCC)(n) hairpin are ''flipped out'' more easily t o the activated state than those in the corresponding Watson-Crick dup lex, (GCC)(n).(GGC)(n) and this makes the hairpin a better target for methylation by the human methyltransferase, the enzyme that methylates the Cs at the CpG sites. (C) 1998 Academic Press.