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.
Mariappan
S V
SV
Theoretical Biology and Biophysics, Los alamos National Laboratory, Los Alamos, NM 87545, USA.
Silks
L A
LA
Bradbury
E M
EM
Gupta
G
G
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