Hairpin induced slippage and hyper-methylation of the fragile X DNA triplets.
The fragile X triplet repeats, (GCC)n x (GGC)n are located at the 5' untranslated region of the FMR-1 gene. Inordinate repeat expansion and hyper-methylation of the CpG islands inside the repeat lead to the suppression of the FMR-1 gene and the subsequent onset and progression of the disease. Previously, we have shown that the (GCC)n strand of the fragile X repeat readily forms hairpin structures under physiological conditions (Chen et al., Proc. Natl. Acad. Sci. USA, 92:5199-5203, 1995: Mariappan et al., Nucl. Acid Res. 24:784-792, 1996). Here, we show by an in vitro assay that formation of the (GCC)n hairpins leads to slippage during replication. The slippage structure is a three-way junction with two Watson-Crick, (GCC)n x (GGC)n, arms and a third (GCC)n hairpin arm. Formation of such slippage structures during replication may explain the observed length polymorphism of the fragile X repeat. We have also studied the substrate efficiency of these three-way junctions toward the human methyltransferase. the enzyme that methylates the CpG sites in DNA. These methylation studies show that the slippage structures induced by the (GCC)n hairpins are 10-15 times more efficient substrates than either the corresponding Watson-Crick duplexes or the (GCC)n hairpins. We demonstrate by appropriate designs that the exceptional substrate efficiency of the three-way junction slippage structures is due to two factors: (i) the presence of the (GCC)n hairpin in which CpG sites are more accessible for methylation than the CpG sites in the Watson-Crick duplex and (ii) the ability of the (GCC)n hairpin in these three-way junctions to move along the Watson-Crick arms that facilitates conversion of low-affinity Watson-Crick CpG sites into high-affinity hairpin CpG sites. Therefore, we suggest that the formation of the (GCC)n hairpins during replication can explain both length polymorphism and hyper-methylation of the fragile X repeats.
Chen
X
X
Theoretical Biology and Biophysics, Los Alamos National Laboratory, NM 87545, USA.
Mariappan
S V
SV
Moyzis
R K
RK
Bradbury
E M
EM
Gupta
G
G
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018911:Trinucleotide Repeats
D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases