Fragile X DNA triplet repeats, (GCC)n, form hairpins with single hydrogen-bonded cytosine.cytosine mispairs at the CpG sites: isotope-edited nuclear magnetic resonance spectroscopy on (GCC)n with selective 15N4-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 hypermethylation associated with the fragile X syndrome. For this, we have performed hetero-nuclear (15N-1H) magnetic resonance spectroscopy to probe the structure of the CpG sites in the (GCC)n hairpins that are 15N-labeled at the amino (N4) groups of specific cytosine bases. Analyses 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 probably form single hydrogen-bonded C.C mispairs. Measurements of pH-dependent 1H 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 the CpG sites of the (GCC)n hairpin are "flipped out" more easily to the activated state than those in the corresponding Watson-Crick duplex, (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.
Theoretical Biology and Biophysics, Los alamos National Laboratory, Los Alamos, NM 87545, USA.
S V
SV
Mariappan
L A
LA
Silks
E M
EM
Bradbury
G
G
Gupta
D020121:5' Untranslated Regions / Q000737:chemistry; D020137:Base Pair Mismatch; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009419:Nerve Tissue Proteins / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D016601:RNA-Binding Proteins; D018911:Trinucleotide Repeats / Q000235:genetics*
D020121:5' Untranslated Regions; D003596:Cytosine; D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human